Lrp4/Corin Dopaminergic Neuron Progenitor Cell Markers

ABSTRACT

The present invention relates to polynucleotide probes and antibodies for detecting Lrp4/Corin dopaminergic neuron progenitor cell markers, which enable the efficient separation of dopaminergic neuron progenitor cells; and methods for selecting the progenitor cells by the use thereof.

TECHNICAL FIELD

The present invention relates to polynucleotide probes and antibodiesfor detecting and selecting dopaminergic neuron progenitor cells, andmethods for detecting and selecting dopaminergic neuron progenitor cellsby using them, and kits and therapeutic methods for treatingneurodegenerative diseases such as Parkinson's disease usingdopaminergic neuron progenitor cells.

BACKGROUND ART

The dopamine system is an extremely important system for essential motorregulation, hormone secretion regulation, emotion regulation, and suchin the mammalian brain. Thus, abnormalities in dopaminergic neuraltransmission cause various neural disorders. For example, Parkinson'sdisease is a neurodegenerative disease of the extrapyramidal system thatoccurs due to specific degeneration of dopaminergic neurons in thesubstantia nigra of the midbrain (Harrison's Principles of InternalMedicine, Vol. 2, 23rd edition, Isselbacher et al., ed., McGraw-HillInc., NY (1994), pp. 2275-7). As a primary therapeutic method forParkinson's disease, oral administration of L-DOPA(3,4-dihydroxyphenylalanine) is performed to compensate for the decreasein the amount of dopamine produced; however, the duration of the effectis known to be unsatisfactory.

More recently, a therapeutic method for Parkinson's disease wasattempted in which the midbrain ventral regions of 6 to 9-week oldaborted fetuses containing dopaminergic neuron progenitor cells aretransplanted to compensate for the loss of dopaminergic neurons (PatentDocument 1; and Non-Patent Documents 1 to 6). However, in addition tocell supply and ethical issues (Rosenstain (1995) Exp. Neurol. 33: 106;Turner et al. (1993) Neurosurg. 33: 1031-7), this method is currentlyunder criticism for various other problems, including risk of infectionand contamination, immunological rejection of transplants (Lopez-Lozanoet al. (1997) Transp. Proc. 29: 977-980; Widner and Brudin (1988) BrainRes. Rev. 13: 287-324), and low survival rates due to the primarydependence of fetal tissues on lipid metabolism rather than glycolysis(Rosenstein (1995) Exp. Neurol. 33: 106).

In order to resolve the ethical issues and shortage of supply, methodshave been proposed that use, for example, porcine cortex, stria, andmidbrain cells (for example, see Patent Documents 2 to 4). In thesemethods, a complex procedure that involves altering cell surfaceantigens (MHC class I antigens) is required to suppress rejection. Amethod involving local immunosuppression by simultaneously transplantingSertoli's cells has been proposed as a method for eliminating transplantrejection (Patent Documents 5 and 6; and Non-Patent Document 7). It ispossible to obtain transplant cells from relatives that have matchingMHCs, bone marrow from other individuals, bone marrow banks, orumbilical cord-blood banks. However, if it were possible to use thepatient's own cells, the problem of rejection reactions could beovercome without any laborious procedures or trouble.

Therefore, as transplant materials, the use of dopaminergic neuronsdifferentiated in vitro from non-neural cells such as embryonic stem(ES) cells and bone marrow interstitial cells, instead of cells fromaborted fetuses, is considered to be promising. In fact, functionaldopaminergic neurons were reported to have been formed by transplantingES cells to lesion stria of a rat Parkinson's disease model (Non-PatentDocument 8). It is believed that the importance of regenerative therapyfrom ES cells or the patient's own nerve stem cells will increase in thefuture.

In treating damaged nerve tissue, it is necessary to reconstruct brainfunction, and in order to form a suitable link with surrounding cells(network formation), it is necessary to transplant immature cells, cellscapable of differentiating into neurons in vivo. In the transplanting ofneuron progenitor cells, in addition to the aforementioned problemregarding supply, there is also the possibility that the progenitorcells will differentiate into groups of heterogeneous cells. Forexample, in treating Parkinson's disease, it is necessary to selectivelytransplant the catecholamine-containing neurons that produce dopamine.Examples of transplant cells that have previously been proposed for usein the treatment of Parkinson's disease include striatum (Non-PatentDocuments 3 and 9), immortalized cell lines derived from human fetalneurons (Patent Documents 7 to 9), human postmitotic neurons derivedfrom NT2Z cells (Patent Document 10), primordial neuron cells (PatentDocument 11), cells and bone marrow stroma cells transfected withexogenous genes so as to produce catecholamines such as dopamines(Patent Documents 12 and 13), and genetically engineered ES cells(Non-Patent Document 8). Additionally, the use of dopaminergic neuronsformed by contacting nerve progenitor cells derived from fetal midbraintissue with FGF-8 and Shh (Patent Document 14), and of tyrosinehydroxylase-expressing cells obtained by treating NT2 nerve cells withretinoic acid (Patent Document 15) has been proposed. However, none ofthese contain only dopaminergic neurons or cells that differentiate intodopaminergic cells.

A method has been proposed for selectively concentrating and isolatingdopaminergic neurons from undifferentiated cell populations. In thismethod, a reporter gene that expresses a fluorescent protein isintroduced into each cell of a cell population, under the control of agene promoter/enhancer such as the tyrosine hydroxylase expressed indopaminergic neurons (hereinbelow, also referred to as “TH”), and thencells emitting fluorescence are isolated.

The dopaminergic neurons are visualized in their viable state, thenconcentrated, isolated, and identified (Patent Document 16). This methodrequires the complicated step of introducing an exogenous gene, andfurther, the presence of a reporter gene poses problems of toxicity andimmunogenicity when used in conjunction with gene therapy.

[Patent Document 1] U.S. Pat. No. 5,690,927[Patent Document 2] Japanese Patent Kohyo Publication No. (JP-A)H10-508487 (unexamined Japanese national phase publication correspondingto a non-Japanese international publication)

[Patent Document 3] JP-A H10-508488 [Patent Document 4] JP-A H10-509034[Patent Document 5] JP-A H11-509170 [Patent Document 6] JP-A H11-501818[Patent Document 7] JP-A H8-509215 [Patent Document 8] JP-A H11-506930[Patent Document 9] JP-A 2002-522070 [Patent Document 10] JP-AH9-5050554 [Patent Document 11] JP-A H11-509729 [Patent Document 12]JP-A 2002-504503 [Patent Document 13] JP-A 2002-513545

[Patent Document 14] U.S. Pat. No. 6,277,820

[Patent Document 15] International Publication WO 00/06700

[Patent Document 16] Japanese Patent Application Kokai Publication No.(JP-A) 2002-51775 (unexamined, published Japanese patent application)[Non-Patent Document 1] Spencer et al. (1992) N. Engl. J. Med. 327:1541-8[Non-Patent Document 2] Freed et al. (1992) N. Engl. J. Med. 327:1549-55[Non-Patent Document 3] Widner et al. (1992) N. Engl. J. Med. 327:1556-63[Non-Patent Document 4] Kordower et al. (1995) N. Engl. J. Med. 332:1118-24

[Non-Patent Document 5] Defer et al. (1996) Brain 119: 41-50

[Non-Patent Document 6] Lopez-Lozano et al. (1997) Transp. Proc. 29:977-80

[Non-Patent Document 7] Selawry and Cameron (1993) Cell Transplant 2:123-9 [Non-Patent Document 8] Kim et al. (2002) Nature 418: 50-56

[Non-Patent Document 9] Lindvall et al. (1989) Arch. Neurol. 46: 615-31

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

One of the major problems in Parkinson's disease transplantation therapyat the moment is that both in vitro differentiated dopaminergic neuronprecursor cells and midbrain ventral region of aborted fetuses aremixtures of a myriad cell types. When considering safety in neuralcircuit formation, it is preferable to use isolated cells that compriseonly the cell type of interest. Furthermore, when considering the riskof tumorigenesis, it is believed better to use isolated postmitoticneurons. Moreover, when considering the survival of cells at theirtransplantation site in the brain, and their ability to properly form anetwork, it is expected that therapeutic effects can be further improvedby isolating dopaminergic neuron progenitor cells at as early a stage aspossible.

Means to Solve the Problems

In order to isolate genes specific to dopaminergic neuron progenitorcells, a gene specifically expressed in the most ventral region of theE12.5 murine midbrain containing dopaminergic neurons was identifiedusing a modification (“Method for Homogenizing the Amounts of DNAFragments and Subtraction Method”, WO 2002/103007 (InternationalPublication Pamphlet)) of the subtraction method (N-RDA:Representational Difference Analysis; RDA (Listsyn N. A. (1995) TrendsGenet. 11: 303-7) by additionally dividing the ventral region into tworegions in the dorsoventral direction. As a result, the presentinventors successfully isolated Lrp4/Corin. Lrp4 encoded a type IItransmembrane protein (FIG. 1).

Lrp4 mRNA is specifically expressed in a ventral midline region in themidbrain. The areas where Lrp4 mRNA is expressed match the areas wheredopaminergic neuron proliferative progenitor cells are present.Furthermore, when Lrp4 expression is compared to that of TH, which is amarker for dopaminergic neurons, their signals are in identicaldorsoventral positions, but they do not overlap (FIGS. 4 and 5). Thisindicated that Lrp4 mRNA is not expressed in these progenitor cells,which had stopped cell division and migrated to the neural tube outerlayer. Therefore, by using Lrp4 mRNA as an index, it is possible tospecifically detect and select dopaminergic neuron proliferativeprogenitor cells.

Thus, the present invention provides dopaminergic neuron proliferativeprogenitor cell marker polynucleotide probes capable of specificallydetecting Lrp4 mRNA, and methods for selecting dopaminergic neuronproliferative progenitor cells utilizing these probes. Furthermore, thepresent invention relates to dopaminergic neuron proliferativeprogenitor cells prior to cell cycle exit, selected by using suchnucleotide probes (hereinbelow, sometimes referred to simply as“dopaminergic neuron proliferative progenitor cells”); as well asmethods for isolating dopaminergic neuron proliferative progenitorcell-specific genes and genes specific for each maturation stage fromprogenitor cell to dopaminergic neuron, utilizing the proliferativeprogenitor cells; and methods of screening for compounds which inducedifferentiation or proliferation of the progenitor cells, usingmaturation as an index. It is also possible to culture the proliferativeprogenitor cells selected by using the nucleotide probes of the presentinvention, and to obtain dopaminergic neuron lineage cells comprisingthe postmitotic dopaminergic neuron precursor cells. The term“dopaminergic neuron lineage cells” used herein refers to dopaminergicneuron proliferative progenitor cells, postmitotic dopaminergic neuronprecursor cells, and/or dopaminergic neurons. The dopaminergic neuronlineage cells can also be utilized for the methods for isolating genesspecific to each stage of maturation to dopaminergic neuron, and themethods of screening for compounds which induce the differentiation orproliferation of progenitor cells, using maturation as an index.Therefore, the present invention relates to methods for obtaining thedopaminergic neuron lineage cells by culturing the dopaminergic neuronproliferative progenitor cells selected by using the nucleotide probesof the present invention; cells obtained in this way; methods forisolating genes specific to each stage of maturation to dopaminergicneurons that use the cells; and methods of screening for compounds whichinduce the differentiation or proliferation of the cells usingmaturation as an index.

Furthermore, the present inventors produced an anti-Lrp4 antibody, andexamined Lrp4 protein expression. First, by analyzing its expression intissues (FIG. 8), the Lrp4 protein was confirmed to be expressed in thesame way as Lrp4 mRNA. In this experiment, Lrp4 protein signals werealso detected in TH-expressing areas. However, since the proliferativeprogenitor cells extend processes toward the outer layer of the neuraltube, this signal could not be determined to be caused by detectingproteins on the processes or the TH-expressing cells also expressed theLrp4 protein. Next, by using the anti-Lrp4 antibody, whether Lrp4protein was expressed on the cell surface was analyzed by flowcytometry. Cells in which Lrp4 mRNA expression was confirmed wereobtained by inducing the differentiation of ES cells into dopaminergicneuron progenitor cells in vitro (SDIA method), and used as samples. Asa result, it was confirmed that the Lrp4 protein was certainly expressedon the surface of the cells (FIG. 9). Such proteins expressed on thecell surface are particularly preferable to use as separation markersbecause living cells can be selected (see FIG. 15). In addition, EScells were induced to differentiate in vitro into the dopaminergicneuron progenitor cells by the 5-stage method, and Lrp4 expression wasconfirmed by RT-PCR and flow cytometry using the anti-Lrp4 monoclonalantibody. As a result, Lrp4 was revealed to also be expressed indopaminergic neuron progenitor cells differentiated by the 5-stagemethod (FIGS. 17A and 17B).

Next, Lrp4-positive cells were separated from cells induced todifferentiate (SDIA method) and from mouse fetal ventral midbrain cellsby a cell sorter using the anti-Lrp4 antibody. Gene expression in theseparated cells was analyzed by the RT-PCR method. As a result,expression of the neuron proliferative progenitor cell marker Nestin wasobserved. In addition, cells expressing MAP2, which is a marker forpostmitotic neurons, were also revealed to be included (FIG. 10). TH andNurrl, which is a marker for postmitotic dopaminergic neuron precursorcells, were expressed at higher levels in an Lrp4-positive cellpopulation than in an Lrp4-negative cell population. Therefore, unlikein the case of using Lrp4 mRNA as the index, when cells are selectedusing antibodies by using Lrp4 protein as an index, it is possible toisolate the dopaminergic neuron progenitor cells, including postmitoticdopaminergic neuron precursor cells. Hereinbelow, the term “dopaminergicneuron progenitor cells” refers to dopaminergic neuron proliferativeprogenitor cells and postmitotic dopaminergic neuron precursor cells.For cells separated using the anti-Lrp4 antibody from those cellsinduced to differentiate from the ES cells in vitro by the SDIA method,the expression of ERas and Nanog expressed specifically in the ES cellswas analyzed. As a result, expression was not observed in Lrp4-positivecells, whereas expression of both genes was observed in Lrp4-negativecells (FIG. 18). Therefore, by selecting cells using the anti-Lrp4antibody, it is possible to select and remove undifferentiated ES cellsafter inducing differentiation. Furthermore, Lrp4-positive cells wereseparated from cell populations including dopaminergic neuron progenitorcells induced to differentiate in vitro from ES cells by the 5-stagemethod. Next, the separated Lrp4-positive cells were cultured in vitroto induce TH protein-positive dopaminergic neurons (FIG. 17C). Thisrevealed that Lrp4-positive cells induced by the 5-stage method aredopaminergic neuron progenitor cells and can mature in vitro.

Accordingly, the present invention provides antibodies whichspecifically detect the Lrp4 protein and methods that utilize theantibodies to select dopaminergic neuron progenitor cells. Furthermore,the present invention relates to dopaminergic neuron progenitor cellsselected by using such antibodies; as well as methods that use theprogenitor cells to isolate dopaminergic neuron progenitor cell-specificgenes and genes specific for each maturation stage from progenitor cellto dopaminergic neuron; and methods of screening for compounds whichinduce the differentiation or proliferation of the progenitor cellsusing maturation as an index. It is also possible to obtain dopaminergicneuron lineage cells at other differentiation stages by culturing theprogenitor cells selected using the antibodies of the present invention.Such cells can also be utilized in the methods for isolating genesspecific to each stage of maturation to dopaminergic neuron, and themethods of screening for compounds which induce the differentiation orproliferation of the progenitor cell using maturation as an index.Accordingly, the present invention also relates to methods for obtainingdopaminergic neuron lineage cells by culturing dopaminergic neuronprogenitor cells selected using the antibodies of the present invention;cells obtained using the methods; methods that use the cells to isolategenes specific to each stage of maturation to dopaminergic neurons; andmethods of screening for compounds which induce the differentiation orproliferation of the cells using maturation as an index.

Furthermore, the present inventors transplanted Lrp4-expressing cellsseparated using the anti-Lrp4 monoclonal antibody into the striatum ofParkinson's disease model mice. As a result, EGFP-positive cells wereobserved in the striatum of transplanted mice (Table 1). Thus it appearsthat the transplanted Lrp4 protein-positive cells were successfullyengrafted in the striatum of the Parkinson's disease model mice. Most ofthe successfully engrafted cells were positive for the mature neuronmarker MAP2, and EGFP-positive axons were observed to extend into thestriatum (Table 1 and FIG. 16). Most of the successfully engrafted cellsdifferentiated into nerve cells and then matured, whereas thetransplanted Lrp4 protein-positive cells were neural progenitor cells,and about 20% of the successfully engrafted cells were TH-positive.These findings strongly suggested that at least some of the transplantedLrp4 protein-positive cells differentiated into dopaminergic neurons.Therefore, the dopaminergic neuron progenitor cells separated accordingto the present invention can differentiate into dopaminergic neuronsupon being transplanted in to the brain, and may be effective for thetreatment of Parkinson's disease. That is, the present invention alsorelates to kits for treating neurodegenerative diseases, preferablyParkinson's disease, comprising the dopaminergic neuron progenitor cellsseparated according to the present invention; and methods for treatingneurodegenerative diseases, preferably Parkinson's disease, comprisingthe step of transplanting dopaminergic neuron progenitor cells into thebrains of patients.

EFFECTS OF THE INVENTION

There have been no previous reports of genes encoding membrane proteinsspecifically expressed in dopaminergic neuron proliferative progenitorcells. Antibodies to Lrp4 protein expressed on the cell membrane surfaceare believed to be extremely effective in isolating dopaminergic neuronprogenitor cells. For example, pure dopaminergic neuron progenitor cellscan be obtained by isolating Lrp4-expressing cells from the ventralmidbrain region or cultured cells containing dopaminergic neuronprogenitor cells differentiated in vitro, using anti-Lrp4 antibodies(FIG. 15). Lrp4 expression was confirmed on both of the dopaminergicneuron progenitor cells induced by two different differentiation methods(SDIA method and 5-stage method). Accordingly, Lrp4 is useful as adopaminergic neuron progenitor cell marker, regardless of cell source.In addition, since ERas and Nanog, which are specifically expressed inES cells, are not expressed in Lrp4-positive cells, it is possible toselect undifferentiated ES cells using Lrp4 expression as an index.

Moreover, in the present invention, the isolated dopaminergic neuronprogenitor cells can also be transplanted directly, or after having beengrown in vitro. The dopaminergic neuron progenitor cells of the presentinvention also have the potential to differentiate and mature at theoptimum region in the brain, as well as the potential to additionallygrow in vivo, and can be expected to demonstrate long-term therapeuticeffects. In addition, if Lrp4-expressing cells are transplanted afterhaving differentiated and matured in vitro, they can be expected todemonstrate therapeutic effects, even if for some reason they do notdifferentiate into dopaminergic neurons in vivo. In consideration of therisks of tumorigenesis and such, an even higher degree of safety can beexpected if cells that have been isolated using a postmitoticdopaminergic neuron precursor cell marker such as 65B13 (WO 2004/038018pamphlet) after differentiating Lrp4-expressing cells grown in vitro aretransplanted. The use of Lrp4-expressing cells for transplantationtherapy after being isolated, regardless of the method, enables a highdegree of safety since only the cell type of interest is isolated. Inaddition, since the earliest dopaminergic neuron progenitor cells can beused, high therapeutic efficacy can be expected in terms of survivalrate, network formation ability, and such. Further, even if the besttherapeutic effects cannot be achieved by these early progenitor cellsimmediately after isolation, since progenitor cells isolated usingmarkers of the present invention can mature in vitro by culturing orsuch, materials in the optimum stage of differentiation can be prepared(FIG. 6).

On the other hand, pure dopaminergic neuron progenitor cells are alsouseful in the search for therapeutic targets for Parkinson's disease,such as for use in the isolation of genes specific to dopaminergicneurons. In particular, dopaminergic neuron proliferative progenitorcells are useful for research on the maturation process of dopaminergicneurons, screening systems using maturation as an index, drug screeningin which progenitor cells are grown in vitro or in vivo, screening fordrugs that induce differentiation of progenitor cells in vivo (in vivoregenerative therapy drugs), and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the structure of Lrp4. TM: transmembranedomain, FRI: frizzeled domain, LDLa: LDL receptor domain, SR: scavengerreceptor domain, Protease: serine protease domain.

FIG. 2 is a set of photographs showing the results of Lrp4 and Shh mRNAexpression analysis in E12.5 mouse ventral hindbrain region and spinalcord by in situ hybridization.

FIG. 3 is a set of photographs showing the results of Lrp4, Shh,tyrosine hydroxylase (TH), and NCAM mRNA expression analysis in E12.5mouse ventral midbrain region by in situ hybridization.

FIG. 4 is a schematic diagram of the Lrp4 expression pattern in themidbrain, and photographs indicating the expression of mRNAs of Lrp4,tyrosine hydroxylase (TH), Sim-1, and NCAM in the ventral midbrain ofE12.5 mice analyzed by in situ hybridization. VZ: ventricular zone; andML: mantle layer.

FIG. 5 is a set of photographs showing the results of Lrp4 mRNAexpression analysis in the E12.5 mouse central nervous system by in situhybridization. A: sagittal cross-section, B: enlarged photograph of thearea inside the box in A, C: cross-section at the location of the redline in A, D: Expression of Lrp4, Shh, and tyrosine hydroxylase (TH)mRNA in the E12.5 mouse midbrain ventral region.

FIG. 6 schematically shows the timing of expression of Lrp4, NCAM, TH,and DAT mRNAs from the generation to maturation of dopaminergic neurons.

FIG. 7 (top panel consisting of a drawing and photograph) schematicallyshows the inhibition of differentiation of ES cells into dopaminergicneurons by the SDIA method. The bottom photograph shows the results ofinvestigating the expression of Lrp4 mRNA in dopaminergic neuronsdifferentiated from ES cells over time, using the SDIA method by RT-PCR.

FIG. 8 is a photograph showing Lrp4 protein expression in the E12.5mouse midbrain.

FIG. 9 shows Lrp4 protein expression on the SDIA-differentiated cellsurface analyzed by flow cytometry using the anti-Lrp4 antibody.

FIG. 10 is a set of photographs showing the expression of variousdopaminergic neuron markers in Lrp4-positive cells analyzed by RT-PCR.

FIG. 11 schematically shows the expression periods of Lrp4 mRNA andprotein, and TH mRNA from the development to maturation of dopaminergicneurons. This shows that both dopaminergic neuron proliferativeprogenitor cells and postmitotic dopaminergic neuron precursor cells arepresent in Lrp4-expressing cells.

FIG. 12 is a set of photographs showing the results of examining thedifferentiation stages of Lrp4-positive cells.

FIG. 13 is a set of photographs showing the in vitro proliferation ofLrp4-positive cells.

FIG. 14 is a set of photographs showing that Lrp4-positive cellsdifferentiate into dopaminergic neurons.

FIG. 15 schematically shows the separation and application ofdopaminergic neuron progenitor cells using anti-Lrp4 antibody.

FIG. 16 is a set of photographs showing the in vivo differentiation oftransplanted Lrp4-positive cells.

FIG. 17 shows a diagram and photographs indicating Lrp4 expression incells differentiated by the 5-stage method, and differentiation ofLrp4-positive cells into dopaminergic neurons. The arrows in panel Cindicate TH protein-positive dopaminergic neurons.

FIG. 18 is a set of photographs showing the results of using RT-PCR toanalyze the expression of ES cell-specific genes (ERas and Nanog) inLrp4-positive cells and Lrp4-negative cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the embodiments of the present invention will be described,but they are exemplified to illustrate the present invention, and not tolimit the present invention to these embodiments. The present inventioncan be carried out in various forms without departing from the scopethereof.

References, publications, patent publications, and patent referencescited herein are incorporated by reference.

<Marker Polynucleotide Probes>

The dopaminergic neuron proliferative progenitor cell markerpolynucleotide probes of the present invention are used as markersand/or reagents for selecting and/or detecting dopaminergic neuronproliferative progenitor cells. Polynucleotides used for this probecomprise a nucleotide sequence complementary to the nucleotide sequenceof SEQ ID NO: 1 or 2 detected in dopaminergic neuron proliferativeprogenitor cells. SEQ ID NO: 1 is the nucleotide sequence of murine Lrp4cDNA. SEQ ID NO: 2 is the nucleotide sequence of human Lrp4 cDNA. Bothsequences are registered in GenBank (murine: Accession No. NM 016869;human: Accession No. XM_(—)035037).

Here, a “marker polynucleotide probe” refers to a polymer composed of anumber of nucleotides, such as deoxyribonucleic acids (DNAs) orribonucleic acids (RNAs), or nucleotide pairs, where the polymer shouldbe able to detect expression of Lrp4, particularly transcribed mRNA.Double-stranded cDNAs are also known to be able to be used as probes intissue in situ hybridization, and such double-stranded cDNAs areincluded in the markers of the present invention. RNA probes(riboprobes) are particularly preferable as marker polynucleotide probesfor detecting RNAs in tissue. If needed, the marker polynucleotideprobes of the present invention can also contain non-naturally-occurringnucleotides such as 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine,2′-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, dihydrouridine,2′-O-methylpseudouridine, β-D-galactosylqueuosine, 2′-O-methylguanosine,inosine, N6-isopentenyladenosine, 1-methyladenosine,1-methylpseudouridine, 1-methylguanosine, 1-methylinosine,2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine,3-methylcytidine, 5-methylcytidine, N6-methyladenosine,7-methylguanosine, 5-methylaminomethyluridine,5-methoxyaminomethyl-2-thiouridine, β-D-mannosylqueuosine,5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine,5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine,N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine,N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine,uridine-5-oxyacetic acid-methyl ester, uridine-5-oxyacetic acid,wybutoxosine, pseudouridine, queuosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine,N-((9-β-D-ribofuranosylpurin-6-yl)carbamoyl)threonine,2′-O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine, and3-(3-amino-3-carboxy propyl)uridine.

Moreover, marker polynucleotide probes of the present invention comprisenucleotide sequences complementary to nucleotide sequences encoding theamino acid sequence of SEQ ID NO: 3 or 4. The nucleotide sequence thatencodes the amino acid sequence of SEQ ID NO: 3 or 4 includes not onlynucleotide sequences of SEQ ID NO: 1 or 2, but also nucleotide sequencesthat differ from the sequences of SEQ ID NO: 1 or 2 due to degeneracy ofthe genetic code. The marker polynucleotide probes of the presentinvention also include those which comprise sequences complementary tonucleotide sequences encoding a sequence that lacks the transmembranedomain in the amino acid sequence of SEQ ID NO: 3 or 4. There is nosignal sequence in the amino acid sequence of SEQ ID NO: 3 or 4. Inmurine Lrp4 (SEQ ID NO: 3), amino acid residues 113-135 form atransmembrane domain, while in human Lrp4 (SEQ ID NO: 4), amino acidresidues 46-68 form a transmembrane domain. The sequences described inSEQ ID NOs: 3 and 4 are respectively registered in GenBank (human Lrp4,XP_(—)035037; murine Lrp4, NP_(—)058565).

Herein, the phrase “complementary to a nucleotide sequence” encompassesnot only cases wherein a nucleotide sequence completely pairs with thetemplate, but also includes those that have at least 70%, preferably80%, more preferably 90%, and even more preferably 95% or more (forexample, 97% or 99%) of the nucleotides paired with the template.Pairing refers to the formation of a chain in which T (U in the case ofRNAs) corresponds to A, A corresponds to T or U, G corresponds to C, andC corresponds to G in the nucleotide sequence of the templatepolynucleotide. Homologies at the nucleotide sequence level betweencertain polynucleotides can be determined by the BLAST algorithm(Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). The BLASTNprogram for nucleotide sequences (Altschul et al. (1990) J. Mol. Biol.215: 403-410) has been developed based on this algorithm, and can beused to determine the homology of marker polynucleotide probe sequences(see http://www.ncbi.nlm.nih.gov for a specific example of analysismethods).

Moreover, marker polynucleotide probes of the present invention includepolynucleotides that contain nucleotide sequences that hybridize understringent conditions with polynucleotides comprised of the nucleotidesequence of SEQ ID NO: 1 or 2. Although polynucleotides with anucleotide sequence indicated in SEQ ID NO: 1 or 2 are known withrespect to Lrp4, alternative isoforms and allelic variants may alsoexist. Polynucleotides with a sequence complementary to such isoformsand allelic variants can also be used as marker polynucleotides of thepresent invention. Such isoforms and allelic variants can be obtainedfrom cDNA libraries or genomic libraries derived from animals such ashumans, mice, rats, rabbits, hamsters, chickens, pigs, cows, goats, andsheep, by using a polynucleotide probe comprising a nucleotide sequenceof SEQ ID NO: 1 or SEQ ID NO: 2, in known hybridization methods such ascolony hybridization, plaque hybridization, or Southern blotting. See“Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring HarborPress (1989)) for methods of cDNA library construction. In addition,commercially available cDNA libraries or genomic libraries may also beused.

More specifically, in constructing a cDNA library, total RNA is firstprepared from cells, organs, tissues, or such that express Lrp4, byknown techniques such as guanidine ultracentrifugation (Chirwin et al.(1979) Biochemistry 18: 5294-5299) or AGPC (Chomczynski and Sacchi(1987) Anal. Biochem. 162: 156-159), followed by purification of mRNAusing an mRNA Purification Kit (Pharmacia), or such. A kit for directmRNA preparation, such as the QuickPrep mRNA Purification Kit(Pharmacia), may also be used. Next, cDNAs are synthesized from theresulting mRNAs using reverse transcriptase. cDNA synthesis kits, suchas the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(Seikagaku Corporation), are also commercially available. Other methodsthat use the 5′-RACE method to synthesize and amplify cDNA by PCR mayalso be used (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002; Belyavsky et al. (1989) Nucleic Acids Res. 17: 2919-32). Inaddition, in order to construct cDNA libraries containing a highpercentage of full-length clones, known techniques such as theoligo-capping method (Maruyama and Sugano (1994) Gene 138: 171-4; Suzuki(1997) Gene 200: 149-56) can also be employed. The cDNA obtained in thismanner can be then incorporated into a suitable vector.

Examples of stringent hybridization conditions suitable for use in thepresent invention include “2×SSC, 0.1% SDS, 50° C.”, “2×SSC, 0.1% SDS,42° C.” and “1×SSC, 0.1% SDS, 37° C.”. Examples of conditions of higherstringency include “2×SSC, 0.1% SDS, 65° C.”, “0.5×SSC, 0.1% SDS, 42°C.” and “0.2×SSC, 0.1% SDS, 65° C.”. More specifically, a method thatuses the Rapid-hyb buffer (Amersham Life Science) can be carried out byperforming pre-hybridization at 68° C. for 30 minutes or more, adding aprobe to allow hybrid formation at 68° C. for one hour or more, washingthree times in 2×SSC/0.1% SDS at room temperature for 20 minutes each,washing three times in 1×SSC/0.1% SDS at 37° C. for 20 minutes each, andfinally washing twice in 1×SSC/0.1% SDS at 50° C. for 20 minutes each.This can also be carried out using, for example, the ExpresshybHybridization Solution (CLONTECH), by performing pre-hybridization at55° C. for 30 minutes or more, adding a labeled probe and incubating at37° C. to 55° C. for one hour or more, washing three times in 2×SSC/0.1%SDS at room temperature for 20 minutes each, and washing once at 37° C.for 20 minutes with 1×SSC/0.1% SDS. Herein, conditions of higherstringency can be achieved by increasing the temperature forpre-hybridization, hybridization, or the second wash. For example,pre-hybridization and hybridization temperatures of 60° C. can be raisedto 68° C. for higher stringency. In addition to factors such as saltconcentration of the buffer and temperature, those with ordinary skillin the art can also integrate other factors, such as probeconcentration, probe length, and reaction time, to obtain Lrp4 isoformsand allelic variants, and corresponding genes derived from otherorganisms.

References such as Molecular Cloning, A Laboratory Manual 2^(nd) ed.(Cold Spring Harbor Press (1989); Section 9.47-9.58), Current Protocolsin Molecular Biology (John Wiley & Sons (1987-1997); Section 6.3-6.4),DNA Cloning 1: Core Techniques, A Practical Approach 2^(nd) ed. (OxfordUniversity (1995); Section 2.10 for conditions, in particular), can bereferred to for detailed information on hybridization procedures.Examples of hybridizing polynucleotides include polynucleotidescontaining a nucleotide sequence that has at least 50% or more,preferably 70%, more preferably 80% and even more preferably 90% (forexample, 95% or more, or 99%) identity with a nucleotide sequencecomprising the nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2. Suchidentities can be determined by the BLAST algorithm (Altschul (1990)Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90: 5873-7) as described in the homologydetermination above. In addition to the above-described BLASTN programfor nucleotide sequences, the BLASTX program for determining theidentity of amino acid sequences (Altschul et al. (1990) J. Mol. Biol.215: 403-10) and the like has been developed based on this algorithm andcan be used (as described above, see http://www.ncbi.nlm.nih.gov. for aspecific example of analysis methods).

Lrp4 isoforms or allelic variants, and other genes with an Lrp4-likestructure or function, can be obtained from cDNA libraries and genomiclibraries of animals such as humans, mice, rats, rabbits, hamsters,chickens, pigs, cows, goats, and sheep, by designing primers based onthe nucleotide sequences of SEQ ID NOs: 1 and 2, using geneamplification technology (PCR) (Current Protocols in Molecular Biology,John Wiley & Sons (1987) Sections 6.1-6.4).

The polynucleotide sequences can be confirmed by using conventionalsequence determination methods. For example, the dideoxynucleotide chaintermination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463) can be used. In addition, sequences can also be analyzed using asuitable DNA sequencer.

Moreover, marker polynucleotide probes of the present invention includethe aforementioned (1) sequences complementary to the nucleotidesequence of SEQ ID NO: 1 or 2, (2) sequences complementary to nucleotidesequences that encode the amino acid sequence described in SEQ ID NO: 3or 4, (3) sequences complementary to nucleotide sequences that encode asequence lacking the transmembrane domain portion of the amino acidsequence described in SEQ ID NO: 3 or 4, and (4) polynucleotidescomprising nucleotide sequences that contain at least 15 consecutivenucleotides in each of the nucleotide sequences that hybridize understringent conditions with a polynucleotide comprised of the nucleotidesequence of SEQ ID NO: 1 or 2. Such polynucleotides comprising anucleotide sequence that contains at least 15 consecutive nucleotidescan be used as a probe for detecting, or as a primer for amplifying, theexpression of Lrp4 mRNA. The nucleotide chain normally consists of 15 to100, and is preferably 15 to 35 nucleotides when used as a probe, or atleast 15 and preferably 30 nucleotides when used as a primer. A primercan be designed to have a restriction enzyme recognition sequence, a tagor such, added to the 5′-end side thereof, and at the 3′ end, a sequencecomplementary to a target sequence. Such polynucleotides, comprising anucleotide sequence that contains at lease 15 consecutive nucleotides,can hybridize with an Lrp4 polynucleotide.

Furthermore, the marker polynucleotide probes of the present inventioncomprise a second polynucleotide which hybridizes under stringentconditions with a first polynucleotide comprising any one of: (1) thenucleotide sequence of SEQ ID NO: 1 or 2; (2) nucleotide sequencescomprising polynucleotides encoding polypeptides comprising the aminoacid sequence of SEQ ID NO: 3 or 4; (3) nucleotide sequences comprisingpolynucleotides encoding polypeptides comprising an amino acid sequencewhich lacks the transmembrane region in the amino acid sequence of SEQID NO: 3 or 4; and (4) nucleotide sequences comprising polynucleotideswhich hybridize under stringent conditions with a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 1 or 2. Polynucleotidesconsisting of a nucleotide sequence comprising at least 15 consecutivenucleotides are preferred as the second polynucleotide.

Marker polynucleotide probes of the present invention can be preparedfrom cells that express Lrp4 by the aforementioned hybridization or PCRor such. In addition, marker polynucleotide probes of the presentinvention can also be produced by chemical synthesis based on known Lrp4sequence data. Riboprobes, which are considered to be particularlypreferable for detecting RNA in tissues, can be obtained by, forexample, inserting a cloned Lrp4 gene or portion thereof into plasmidvector pSP64 in the reverse direction, followed by run-off transcriptionof the inserted sequence portion. Although pSP64 contains an SP6promoter, methods for producing riboprobes by combining phage T3, T7promoter and RNA polymerase are also known. The marker polynucleotideprobes of the present invention may be used as reagents fordiscriminating the dopaminergic neuron proliferative progenitor cells.In the above-described reagents, the polynucleotides as activeingredients may be mixed as necessary with, for example, sterile water,saline, vegetable oils, surfactants, lipids, solubilizers, buffers,stabilizers, and preservatives.

<Antibodies>

The present invention provides dopaminergic neuron progenitor cellmarker antibodies (hereinbelow, sometimes referred to as “antibodies ofthe present invention”) which can be used to select dopaminergic neuronprogenitor cells from brain tissue or cultured cells. Unlike Lrp4 mRNA,the Lrp4 polypeptide is expressed not only in the dopaminergic neuronproliferative progenitor cells prior to cell cycle exit, but also inpostmitotic dopaminergic neuron precursor cells. Therefore, by using theantibodies of the present invention against the polypeptide, it ispossible to select and/or obtain dopaminergic neuron progenitor cellsprior to and after cell cycle exit. Antibodies of the present inventioninclude polyclonal antibodies, monoclonal antibodies, chimericantibodies, single-chain antibodies (scFV) (Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85: 5879-83; The Pharmacology of MonoclonalAntibody, vol. 113, Rosenburg and Moore ed., Springer Verlag (1994) pp.269-315), humanized antibodies, multispecific antibodies (LeDoussal etal. (1992) Int. J. Cancer Suppl. 7: 58-62; Paulus (1985) Behring Inst.Mitt. 78: 118-32; Millstein and Cuello (1983) Nature 305: 537-9;Zimmermann (1986) Rev. Physiol. Biochem. Pharmacol. 105: 176-260; VanDijk et al. (1989) Int. J. Cancer 43: 944-9), and antibody fragmentssuch as Fab, Fab′, F(ab′)2, and Fv. Moreover, antibodies of the presentinvention may also be modified by PEG and such, as necessary. Antibodiesof the present invention may also be produced in the form of a fusionprotein with β-galactosidase, maltose-binding protein, GST, greenfluorescent protein (GFP) and such, to allow detection without the useof a secondary antibody. In addition, antibodies of the presentinvention may be modified by labeling with biotin or such, to allowrecovery using avidin, streptoavidin, or such.

The antibodies of present invention are specific to any of (1)polypeptides encoded by the nucleotide sequence of SEQ ID NO: 1 or 2,(2) polypeptides comprised of the amino acid sequence described in SEQID NO: 3 or 4, (3) polypeptides comprised of an amino acid sequencelacking the transmembrane domain in the amino acid sequence described inSEQ ID NO: 3 or 4, (4) polypeptides comprised of an amino acid sequencewherein one or more amino acids in the amino acid sequence of SEQ ID NO:3 or 4 are deleted, inserted, substituted, or added, (5) polypeptidesencoded by a nucleotide sequence that hybridizes under stringentconditions with a sequence complementary to the nucleotide sequence ofSEQ ID NO: 1 or 2, and (6) polypeptides that are fragments of thepolypeptides of (1) to (5) above, with at least eight amino acidresidues.

Also, the antibodies of the present invention may be antibodies thatbind to polypeptides comprising any one of the following amino acidsequences (1) to (4) or parts thereof: (1) the amino acid sequence ofSEQ ID NO: 3 or 4; (2) amino acid sequences which lack the transmembraneregion in the amino acid sequence of SEQ ID NO: 3 or 4; (3) amino acidsequences mutated by one or more amino acid deletions, substitutions, oradditions, or combinations thereof, in the amino acid sequence of SEQ IDNO: 3 or 4; and (4) amino acid sequences comprising polypeptides encodedby polynucleotides which hybridize under stringent conditions withpolynucleotides comprising a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 1 or 2. The above-describedpolypeptides consisting of partial sequences include polypeptidescomprising preferably at least six consecutive amino acid residues(e.g., 8, 10, 12, or 15 amino acid residues or more).

The amino acid sequences mutated by one or more amino acid deletions,substitutions, or additions, or combinations thereof in the amino acidsequence of SEQ ID NO: 3 include, for example: (i) amino acid sequenceswith 1 to 9 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2,and still more preferably 1) amino acid deletions in the amino acidsequence of SEQ ID NO: 3; (ii) amino acid sequences with 1 to 9 (e.g., 1to 5, preferably 1 to 3, more preferably 1 to 2, and still morepreferably 1) amino acid additions in the amino acid sequence of SEQ IDNO: 3; (iii) amino acid sequences with 1 to 9 (e.g., 1 to 5, preferably1 to 3, more preferably 1 to 2, and still more preferably 1) amino acidssubstituted by other amino acids in the amino acid sequence of SEQ IDNO: 3; and (iv) an amino acid sequence mutated by any combination of theabove (i) to (iii).

The amino acid sequences mutated by one or more amino acid deletions,substitutions, or additions, or any combinations thereof in the aminoacid sequence of SEQ ID NO: 4 include, for example: (i) amino acidsequences with 1 to 9 (e.g., 1 to 5, preferably 1 to 3, more preferably1 to 2, and still more preferably 1) amino acid deletions in the aminoacid sequence of SEQ ID NO: 4; (ii) amino acid sequences with 1 to 9(e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and still morepreferably 1) amino acid additions in the amino acid sequence of SEQ IDNO: 4; (iii) amino acid sequences with 1 to 9 (e.g., 1 to 5, preferably1 to 3, more preferably 1 to 2, and still more preferably 1) amino acidssubstituted by other amino acids in the amino acid sequence of SEQ IDNO: 4; and (iv) amino acid sequences mutated by any combination of theabove (i) to (iii).

The term “amino acid deletions” herein means variants in which one ormore amino acid residues in the sequence are deleted. The term“deletion” includes the deletion of an amino acid at either end of anamino acid sequence, and the deletion of an amino acid in an amino acidsequence.

The term “amino acid additions” herein means variants in which one ormore amino acid residues have been added to the sequence. The term“addition” includes the addition of an amino acid to either end of anamino acid sequence and the addition of an amino acid in an amino acidsequence.

The term “amino acid substitutions” herein means variants in which oneor more amino acid residues in the sequence are substituted withdifferent amino acid residues. When amino acid sequences are modified bysuch substitution, conservative substitution is preferably carried out.The term “conservative substitution” means changing the sequence toencode an amino acid with similar properties to the amino acid prior tosubstitution. Amino acids can be classified according to theirproperties into, for example: non-polar amino acids (Ala, Ile, Leu, Met,Phe, Pro, Trp, and Val); non-charged amino acids (Asn, Cys, Gln, Gly,Ser, Thr, and Tyr); acidic amino acids (Asp and Glu); basic amino acids(Arg, His, and Lys); neutral amino acids (Ala, Asn, Cys, Gln, Gly, Ile,Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val); aliphatic amino acids(Ala and Gly); branched amino acids (Ile, Leu, and Val), hydroxyaminoacids (Ser and Thr); amide-type amino acids (Gln and Asn);sulfur-containing amino acids (Cys and Met); aromatic amino acids (His,Phe, Trp, and Tyr); heterocyclic amino acids (His and Trp); and iminoacids (Pro and 4Hyp).

Therefore, it is preferable that non-polar amino acids are substitutedwith each other, or that non-charged amino acids are substituted witheach other. Of these, substitutions among Ala, Val, Leu, and Ile,between Ser and Thr, between Asp and Glu, between Asn and Gln, betweenLys and Arg, and between Phe and Tyr are preferable for retainingproperties of the proteins. The number and sites of mutated amino acidsare not particularly limited.

As the antibodies of the present invention, the two anti-Lrp4 antibodiesused in Example 4, and modifications comprising fragments thereof, areparticularly preferable. The two antibodies have been internationallydeposited under the following accession numbers:

(1) Name and address of the depositary institution

Name: International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology Address: Central 6, 1-1-1Higashi, Tsukuba-shi, Ibaraki Prefecture, Japan 305-8566

(2) Deposit date: Jul. 14, 2004(3) Accession Nos: FERM BP-10315 and FERM BP-10316 (transferred fromFERM P-20120 and FERM P-20121 domestically deposited in Japan)

Antibodies of the present invention can be produced using a sensitizingantigen such as an Lrp4 polypeptide, or fragments thereof, or cells thatexpress Lrp4 polypeptide or Lrp4 polypeptide fragments. In addition,short Lrp4 polypeptide fragments may also be used as immunogens bycoupling with a carrier such as bovine serum albumin, Keyhole-limpethemocyanin, and ovalbumin. In addition, the Lrp4 polypeptide, orfragments thereof, may be used in combination with known adjuvants, suchas aluminum adjuvant, Freund's complete (or incomplete) adjuvant, orpertussis adjuvant, to enhance immune response to the antigen.

The “Lrp4 polypeptide” in the present invention is a peptide polymer, apreferred example of which is a protein having the amino acid sequencedescribed in SEQ ID NO: 3 or 4. The amino acid residues that compose anLrp4 polypeptide may be naturally occurring or modified. Moreover, theLrp4 polypeptides include proteins lacking a transmembrane domainportion, and fusion proteins modified by other peptide sequences.

In the present invention, the Lrp4 polypeptides should have theantigenicity of the Lrp4 polypeptide, and include polypeptides with anamino acid sequence wherein one or more amino acids in the amino acidsequence of SEQ ID NO: 3 or 4 are deleted, inserted, substituted, oradded. It is well known that mutant polypeptides comprising an aminoacid sequence in which one or more amino acids are deleted, inserted,substituted, or added, maintain the same biological activity as theoriginal polypeptide (Mark et al. (1984) Proc. Natl. Acad. Sci. USA 81:5662-6; Zoller and Smith (1982) Nucleic Acids Res. 10: 6487-500; Wang etal. (1984) Science 224: 1431-3; Dalbadie-McFarland et al. (1982) Proc.Natl. Acad. Sci. USA 79: 6409-13). Such polypeptides that maintain theantigenicity of Lrp4 and have an amino acid sequence in which one ormore amino acids are deleted, inserted, substituted, or added to theamino acid sequence of SEQ ID NO: 3 or 4, can be obtained by preparingpolynucleotides that encode the polypeptides using known methods such assite-directed mutagenesis described in “Molecular Cloning, A LaboratoryManual 2nd ed.” (Cold Spring Harbor Press (1989)), “Current Protocols inMolecular Biology” (John Wiley & Sons (1987-1997); especially section8.1-8.5), Hashimoto-Goto et al. (1995) Gene 152: 271-5, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82: 488-92, Kramer and Fritz (1987) Method.Enzymol. 154: 350-67, Kunkel (1988) Method. Enzymol. 85: 2763-6), andothers, and then suitably expressing. In addition to the above-describedsite-directed mutagenesis, methods for subjecting genes to mutagens canbe used. Alternatively, methods for selectively cleaving genes;removing, adding, or substituting selected nucleotides; and thenligating the genes can also be used.

Lrp4 polypeptide fragments are identical to a portion of theaforementioned Lrp4 polypeptide, and preferably consists of at least sixcontinuous amino acid residues or more (for example, 8, 10, 12, or 15amino acid residues or more). A particularly preferred fragment can beexemplified by a polypeptide fragment lacking an amino terminus,carboxyl terminus, and transmembrane domain. The Lrp4 polypeptidefragments include fragments containing an α-helix and α-helix formingregion, α-amphipathic region, β-sheet and β-sheet forming region,β-amphipathic region, substrate binding region, high antigen indexregion, coil and coil forming region, hydrophilic region, hydrophobicregion, turn and turn forming region, and surface forming region. In thecontext of the present invention, an Lrp4 polypeptide fragment may beany fragment, so long as it has the antigenicity of an Lrp4 polypeptide.The antigen-determining site of a polypeptide can be predicted by usingmethods for analyzing the hydrophobicity/hydrophilicity of an amino acidsequence of a protein (Kyte-Doolittle (1982) J. Mol. Biol. 157: 105-22),or methods of secondary structure analysis (Chou-Fasman (1978) Ann. Rev.Biochem. 47: 251-76), and can be confirmed using computer programs(Anal. Biochem. 151: 540-6 (1985)), or the PEPSCAN method in which ashort peptide is synthesized followed by confirmation of itsantigenicity (Published Japanese Translation of InternationalPublication No. Sho 60-500684), or the like.

Lrp4 polypeptides and Lrp4 polypeptide fragments can be isolated fromLrp4-expressing cells, tissues, etc., based on their physical propertiesand such. In addition, these polypeptides and polypeptide fragments canalso be produced using known genetic recombination techniques orchemical synthesis methods. For example, for in vitro Lrp4 polypeptideproduction, Lrp4 polypeptides can be produced in an in vitro cell-freesystem using methods such as in vitro translation (Dasso and Jackson(1989) Nucleic Acids Res. 17: 3129-44). In contrast, when producingpolypeptides using cells, a polynucleotide that encodes a polypeptide ofinterest is first incorporated into an appropriate vector, a suitablehost cell is selected, and then the cells are transformed by the vector.Subsequently, the transformed cells can be cultured to obtain thepolypeptide of interest.

Appropriate vectors include various vectors such as plasmids, cosmids,viruses, bacteriophages, cloning vectors, and expression vectors(Molecular Cloning, A Laboratory Manual 2^(nd) ed., Cold Spring HarborPress (1989); Current Protocols in Molecular Biology, John Wiley & Sons(1987)). The vectors comprise regulatory sequences for the expression ofa desired polynucleotide in transfected host cells, and thepolynucleotide is incorporated therein so that it will be under thecontrol of the regulatory sequences. Here, the phrase “regulatorysequence” includes promoters, ribosome binding sites, and terminators inthe case of a prokaryotic host cell, and promoters and terminators inthe case of a eukaryotic host cell, and in some cases, may also containtransactivators, transcription factors, poly A signals which stabilizetranscription products, splicing and polyadenylation signals, andothers. Such regulatory sequences comprise all the components requiredfor the expression of a polynucleotide linked thereto. Vectors mayfurther comprise a selection marker. Moreover, a signal peptide requiredfor transferring an intracellularlly expressed polypeptide into thelumen of the endoplasmic reticulum, or the periplasm or extracellularspace when the host is a Gram negative microbe, can also be incorporatedinto an expression vector by linking to a polypeptide of interest. Suchsignal peptides can be signal peptides derived from heterogeneousproteins. Moreover, a linker may be added, and a start (ATG) or stopcodon (TAA, TAG, or TGA) may be inserted as necessary.

Examples of vectors capable of expressing polypeptides in vitro includepBEST (Promega). In addition, various vectors are known to be suitablefor expression in prokaryotic hosts (see, e.g., “Basic MicrobiologyCourse 8—Genetic Engineering” (Kyoritsu Publishing)). When selectingprokaryotic cells as the host, a person with ordinary skill in the artcan suitably select a vector suitable for the host and a method suitablefor introducing the vector into the host. Other examples of hosts thatcan be used to express Lrp4 polypeptides and their antigenic fragmentsinclude fungal cells such as yeasts, higher plants, insects, fish,amphibians, reptiles, birds, mammals, cultured cells (COS, Hela, C127,3T3, BHK, HEK293, Bowes melanoma cells), myeloma, Vero, Namalwa, NamalwaKJM-1, and HBT5637 (Unexamined Published Japanese Patent Application No.Sho 63-299). Vector systems suitable for each cell and methods forintroducing a vector into host cells are also known. Moreover, methodsfor expressing exogenous proteins in animals in vivo (see, e.g., Susumu(1985) Nature 315: 592-4; Lubon (1998) Biotechnol. Annu. Rev. 4: 1-54)and in plant bodies are also known, and can be used to express Lrp4polynucleotides.

DNAs can be inserted into vectors in a ligase reaction using restrictionenzyme sites (Current Protocols in Molecular Biology, John Wiley & Sons(1987) Section 11.4-11.11; Molecular Cloning, A Laboratory Manual 2nded., Cold Spring Harbor Press (1989) Section 5.61-5.63). In addition, anLrp4 polypeptide-encoding expression vector can be designed as necessaryby selecting a nucleotide sequence that has high expression efficiencyin view of the host's codon usage frequency (Grantham et al. (1981)Nucleic Acids Res. 9: r43-74). A host that produces an Lrp4 polypeptidecomprises in its cells a polynucleotide that encodes an Lrp4polypeptide. So long as the polynucleotide does not exist at a naturallyoccurring position in the genome of a host cell, the polynucleotideitself may be regulated by its own promoter, incorporated in the hostgenome, or maintained as an extrachromosomal structure.

Transduction (transformation/transfection) of host cells with vectorscan be performed using well-known methods.

Transformation can be performed, for example, by the method by Cohen etal. (Proc. Natl. Acad. Sci. USA 69, 2110 (1972)), protoplast methods(Mol. Gen. Genet., 168, 111 (1979)), and the competent method (J. Mol.Biol., 56, 209 (1971)) for bacteria (E. coli, Bacillus subtilis, etc.);by the method by Hinnen et al. (Proc. Natl. Acad. Sci. USA 75, 1927(1978)) and lithium methods (J. Bacteriol., 153, 163 (1983)) forSaccharomyces cerevisiae; by leaf disk methods (Science, 227, 129(1985)), electroporation methods (Nature, 319, 791 (1986)), andAgrobacterium methods (Horsch et al., Science, 227, 129 (1985); and Hieiet al., Plant J., 6, 271-282 (1994)) for plant cells; by the method byGraham (Virology, 52, 456 (1973)) for animal cells; and by the method bySummers et al. (Mol. Cell. Biol., 3, 2156-2165 (1983)) for insect cells.

Culturing of host cells is carried out using known methods that areappropriate for the host cell selected. For example, when animal cellsare selected, culturing can be carried out at a pH of about 6 to 8 and atemperature of 30° C. to 40° C. for about 15 to 200 hours, using amedium such as DMEM (Virology 8: 396 (1959)), MEM (Science 122: 501(1952)), RPMI1640 (J. Am. Med. Assoc. 199: 519 (1967)), 199 (Proc. Soc.Biol. Med. 73: 1 (1950)), or IMDM, and adding serum such as fetal calfserum (FCS), as necessary. In addition, the medium may be replaced,aerated, or stirred during the course of culturing, as necessary.

Normally, an Lrp4 polypeptide produced by gene recombination techniquescan be recovered from the medium if the polypeptide is secreted outsideof a cell, or from the body fluid of a transgenic organism. When apolypeptide is produced inside of a cell, the cells can be dissolved andthe polypeptide is recovered from the dissolved product. The polypeptideof interest can be then purified by suitably combining known methods ofprotein purification, such as salting out, distillation, various typesof chromatography, gel electrophoresis, gel filtration, ultrafiltration,recrystallization, acid extraction, dialysis, immunoprecipitation,solvent precipitation, solvent extraction, and ammonium sulfate orethanol precipitation. Examples of chromatographies include ion exchangechromatography, such as anion or cation exchange chromatography,affinity chromatography, reversed-phase chromatography, adsorptionchromatography, gel filtration chromatography, hydrophobicchromatography, hydroxyapatite chromatography, phosphocellulosechromatography, and lectin chromatography (Strategies for ProteinPurification and Characterization: A Laboratory Course Manual, Marshaket al. ed., Cold Spring Harbor Laboratory Press (1996)). Chromatographycan be carried out using a liquid phase chromatography, such as HPLC orFPLC. In addition, for example, a protein fused with GST can be purifiedusing a glutathione column, and a protein with a histidine tag can bepurified using a nickel column. When an Lrp4 polypeptide is produced asa fusion protein, unnecessary portions can be removed using thrombin,factor Xa, or the like, following purification as necessary.

In addition, naturally-occurring polypeptides can also be purified andobtained. For example, polypeptides can be purified by affinitychromatography using antibodies against the Lrp4 polypeptides (CurrentProtocols in Molecular Biology, John Wiley & Sons (1987) Section16.1-16.19). Moreover, purified polypeptides can also be modified usingenzymes, such as chymotrypsin, glucosidase, trypsin, protein kinase, andlysyl endopeptidase, as necessary. In addition to the aforementionedsynthesis and genetic engineering techniques as used for Lrp4polypeptides, Lrp4 polypeptide fragments can also be produced bycleaving an Lrp4 polypeptide, using suitable enzymes, such aspeptidases.

Polyclonal antibodies for selecting dopaminergic neuron progenitor cellscan be obtained from, for example, the serum of an immunized animalafter immunizing a mammal with an Lrp4 polypeptide purified as describedabove, or a fragment thereof, coupled to a desired adjuvant. Althoughthere are no particular limitations on the mammals used, typicalexamples include rodents, lagomorphs, and primates. Specific examplesinclude rodents such as mice, rats, and hamsters, lagomorphs such asrabbits, and primates such as monkeys, including cynomolgus monkeys,rhesus monkeys, baboons, and chimpanzees. Animal immunization is carriedout by suitably diluting and suspending a sensitizing antigen inphosphate-buffered saline (PBS) or physiological saline, mixing with anadjuvant as necessary until emulsified, and injecting into an animalintraperitoneally or subcutaneously. The sensitizing antigen mixed withFreund's incomplete adjuvant is preferably administered several times,every 4 to 21 days. Antibody production can be confirmed by measuringthe level of an antibody of interest in the serum using conventionalmethods. Finally, the serum itself may be used as a polyclonal antibody,or it may be further purified. See, for example, “Current Protocols inMolecular Biology” (John Wiley & Sons (1987) Sections 11.12-11.13), forspecific methods.

A monoclonal antibody can be produced by removing the spleen from ananimal immunized in the manner described above, separating immunocytesfrom the spleen, and fusing with a suitable myeloma cell usingpolyethylene glycol (PEG) or such to establish hybridomas. Cell fusioncan be carried out according to the Milstein method (Galfre and Milstein(1981) Methods Enzymol. 73: 3-46). Here, suitable myeloma cells areexemplified particularly by cells that allow chemical selection of fusedcells. When using such myeloma cells, fused hybridomas can be selectedby culturing in a culture medium (HAT culture medium) that containshypoxanthine, aminopterin, and thymidine, which destroys cells otherthan fused cells. Next, clones that produce antibodies againstpolypeptides of the present invention, or a fragment thereof, can beselected from the established hybridomas. Subsequently, the selectedclone is introduced into the abdominal cavity of a mouse or such, andascites can be collected to obtain a monoclonal antibody. See also“Current Protocols in Molecular Biology” (John Wiley & Sons (1987)Section 11.4-11.11), for information on specific methods. Preferablehybridomas of the present invention can be FERM BP-10315 and FERMBP-10316.

Hybridomas can also be obtained by first sensitizing human lymphocytesthat have been infected by EB virus with an immunogen in vitro, andfusing the sensitized lymphocytes with human myeloma cells (such asU266) to obtain hybridomas that produce human antibodies (UnexaminedPublished Japanese Patent Application No. Sho 63-17688). In addition,human antibodies can also be obtained by using antibody-producing cellsgenerated by sensitizing a transgenic animal with a human antibody generepertoire (WO92/03918; WO93-02227; WO94/02602; WO94/25585; WO96/33735;WO96/34096; Mendez et al. (1997) Nat. Genet. 15: 146-156, etc.). Methodsthat do not use hybridomas can be exemplified by a method in which acancer gene is introduced to immortalize immunocytes such asantibody-producing lymphocytes.

In addition, antibodies of the present invention can also be produced bygenetic recombination techniques (see Borrebaeck and Larrick (1990)Therapeutic Monoclonal Antibodies, MacMillan Publishers Ltd., UK).First, a gene that encodes an antibody is cloned from hybridomas orantibody-producing cells (such as sensitized lymphocytes). The resultinggene is then inserted into a suitable vector, the vector is introducedinto a host, and the host is then cultured to produce the antibody. Thistype of recombinant antibody is also included in the antibodies of thepresent invention. Typical examples of recombinant antibodies includechimeric antibodies, comprising a non-human antibody-derived variableregion and a human antibody-derived constant region, and humanizedantibodies, comprising a non-human-derived antibody complementaritydetermining region (CDR), human antibody-derived framework region (FR),and human antibody constant region (Jones et al. (1986) Nature 321:522-5; Reichmann et al. (1988) Nature 332: 323-9; Presta (1992) Curr.Op. Struct. Biol. 2: 593-6; Methods Enzymol. 203: 99-121 (1991)).

Antibody fragments can be produced by treating the aforementionedpolyclonal or monoclonal antibodies with enzymes such as papain orpepsin. Alternatively, antibody fragments can be produced by geneticengineering techniques using genes that encode antibody fragments (seeCo et al., (1994) J. Immunol. 152: 2968-76; Better and Horwitz (1989)Methods Enzymol. 178: 476-96; Pluckthun and Skerra (1989) MethodsEnzymol. 178: 497-515; Lamoyi (1986) Methods Enzymol. 121: 652-63;Rousseaux et al. (1986) 121: 663-9; Bird and Walker (1991) TrendsBiotechnol. 9: 132-7).

Multispecific antibodies include bispecific antibodies (BsAb), diabodies(Db), and such. Multispecific antibodies can be produced by methods suchas (1) chemically coupling antibodies having different specificitieswith different types of bifunctional linkers (Paulus (1985) BehringInst. Mill. 78: 118-32), (2) fusing hybridomas that secrete differentmonoclonal antibodies (Millstein and Cuello (1983) Nature 305: 537-9),or (3) transfecting eukaryotic cell expression systems, such as mousemyeloma cells, with a light chain gene and a heavy chain gene ofdifferent monoclonal antibodies (four types of DNA), followed by theisolation of a bispecific monovalent portion (Zimmermann (1986) Rev.Physio. Biochem. Pharmacol. 105:176-260; Van Dijk et al. (1989) Int. J.Cancer 43: 944-9). Alternatively, diabodies are dimer antibody fragmentscomprising two bivalent polypeptide chains that can be constructed bygene fusion. These can be produced using known methods (see Holliger etal. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-8; EP404097; WO93/11161).

Recovery and purification of antibodies and antibody fragments can becarried out using Protein A and Protein Q or according to the proteinpurification techniques described above in producing non-antibodypolypeptides (Antibodies: A Laboratory Manual, Ed Harlow and David Lane,Cold Spring Harbor Laboratory (1988)). For example, when using Protein Ato purify antibodies of the present invention, Protein A columns such asHyper D, POROS, or Sepharose F.F. (Pharmacia), can be used. Theconcentration of the resulting antibodies can be determined by measuringabsorbance or by enzyme linked immunosorbent assay (ELISA).

The antigen binding activity of an antibody can be determined usingabsorbance measurements, or by using fluorescent antibody methods,enzyme immunoassay (EIA) methods, radioimmunoassay (RIA) methods, orELISA. When ELISA is used, for example, antibodies of the presentinvention are first immobilized onto a support, such as a plate. An Lrp4polypeptide is added, and then a sample containing the antibody ofinterest is added. Herein, samples containing an antibody of interestinclude, for example, culture supernatants of antibody-producing cells,purified antibodies, and such. Next, secondary antibodies that recognizethe antibodies of the present invention are added, and the plate isincubated. The plate is then washed and a label attached to thesecondary antibody is detected. Namely, if a secondary antibody islabeled with alkaline phosphatase, antigen binding activity can bedetermined by adding an enzyme substrate such as p-nitrophenylphosphate, and measuring absorbance. In addition, a commerciallyavailable system such as BIAcore (Pharmacia) can also be used toevaluate antibody activities.

The present invention relates to reagents which comprise the anti-Lrp4antibody as an active ingredient for identifying dopaminergic neuronprogenitor cells. In the above-described reagents, the antibodies of thepresent invention as active ingredients may be mixed as necessary with,for example, sterile water, saline, vegetable oils, surfactants, lipids,solubilizer, buffers, protein stabilizers (such as BSA and gelatin), andpreservatives.

<Methods for Selecting Dopaminergic Neuron Progenitor Cells and Such>

The present invention provides methods for selecting dopaminergic neuronproliferative progenitor cells as a selectively uniform population. Thedopaminergic neuron proliferative progenitor cells can be selected byusing the marker polynucleotide probes of the present invention. Thepresent invention also provides methods for selecting the dopaminergicneuron progenitor cells as a selectively uniform population. Thedopaminergic neuron progenitor cells can suitably be selected using theantibodies of the present invention. As described above, by using thepolynucleotide probes or antibodies of the present invention, thedopaminergic neuron lineage cells which eventually differentiate intodopaminergic neurons can be specifically selected.

Here, the term “selected” includes both detecting the presence of cellsexpressing markers in a sample, and subsequently separating or isolatingthose progenitor cells after detecting their presence. The presentinvention provides methods for selecting dopaminergic neuronproliferative progenitor cells, comprising a step of contacting markerpolynucleotide probes of the present invention with cell samplescontaining dopaminergic neuron proliferative progenitor cells. In thismethod, marker polynucleotide probes are preferably labeled with aradioactive isotope or non-radioactive compound. Examples of theradioactive isotopes used for labeling include ³⁵S and ³H. When using aradiolabeled marker polynucleotide probe, RNA that binds with the markercan be detected by detecting silver particles using emulsionautoradiography. Examples of non-radioactive isotopes for labeling amarker polynucleotide probe include biotin and digoxigenin. Abiotin-labeled marker can be detected using, for example, avidin labeledwith fluorescence or an enzyme such as alkaline phosphatase orhorseradish peroxidase. On the other hand, anti-digoxigenin antibodieslabeled with fluorescence or an enzyme, such as alkaline phosphatase orhorseradish peroxidase, can be used to detect a digoxigenin-labeledmarker. When using enzyme labeling, detection can be carried out byincubating with the enzyme substrate to form a stable pigment at thelocation of the marker. Fluorescent in situ hybridization (FISH) isconvenient and particularly preferable.

In addition, the present invention provides methods for selectingdopaminergic neuron progenitor cells comprising the step of contactingantibodies for selecting the dopaminergic neuron progenitor cells of thepresent invention with cell samples containing dopaminergic neuronprogenitor cells. Namely, dopaminergic neuron progenitor cells can beacquired by contacting cell samples containing potential dopaminergicneuron progenitor cells with antibodies of the present invention, andselecting those cells that have bound to the antibody (see FIG. 13). Theantibodies of the present invention may also be immobilized on asuitable support, prior to contact with cell samples. Alternatively,cells that bind with the antibodies can be selectively recovered bycontacting cell samples with the antibodies of the present invention,allowing them to bind, and then purifying the antibody by affinitychromatography. For example, if the antibodies of the present inventionare conjugated to biotin, they can be purified on a plate or columnbound with avidin or streptoavidin. In addition, magnetic particles canbe bound to an antibody, for example, and the antibody and cells thatexpress Lrp4 on their surfaces, where the Lrp4 is bound to the antibody,can be recovered using a magnet. Dopaminergic neuron progenitor cellsthat express Lrp4 can be selected by flow cytometry using a cell sorterand fluorescent-labeled anti-Lrp4 antibodies and such.

The dopaminergic neuron progenitor cell populations obtained asdescribed above are cell populations comprising, for example, 40% ormore, preferably 50% or more, more preferably 60% or more, andparticularly preferably 70% or more dopaminergic neuron progenitorcells.

The present invention provides methods for selecting and/or producingpostmitotic dopaminergic neuron precursor cells, which comprise the stepof culturing the dopaminergic neuron progenitor cells selected by usingthe marker polynucleotide probes or antibodies of the present invention.The present invention also provides methods for selecting and/orproducing postmitotic dopaminergic neuron precursor cells, whichcomprise the steps of contacting cell samples comprising thedopaminergic neuron progenitor cells with the marker polynucleotideprobes or antibodies of the present invention; selecting thedopaminergic neuron progenitor cells; and culturing the selected cells.

Furthermore, according to the present invention, the postmitoticdopaminergic neuron precursor cells, which have a low risk oftumorigenesis and are suitable for transplant therapy, can be obtainedby culturing the dopaminergic neuron progenitor cells selected by usingthe marker polynucleotide probes or antibodies of the present invention;and selecting and/or screening by using the postmitotic dopaminergicneuron precursorcell markers. The postmitotic dopaminergic neuronprecursor cell markers can include, for example, 65B13, Nurrl, and TH(WO 2004/038018; Kawasaki et al., Neuron 28:3140 (2000); Wallen et al.,Exp. Cell Res., 253:73746 (1999)). For example, the postmitoticdopaminergic neuron precursor cells can be selected and/or produced bycontacting cell samples comprising dopaminergic neuron progenitor cellswith the marker polynucleotide probes or antibodies of the presentinvention; selecting the dopaminergic neuron progenitor cells; andcontacting the antibody against 65B13 polypeptide with the dopaminergicneuron progenitor cells further cultured as necessary to select thecells expressing the 65B13 polypeptide.

The present invention provides methods for selecting and/or producingdopaminergic neurons, where the methods comprise the step of culturingthe dopaminergic neuron progenitor cells selected by using the markerpolynucleotide probes or antibodies of the present invention. Thepresent invention also provides methods for selecting and/or producingdopaminergic neurons which comprise the steps of contacting cell samplescontaining the dopaminergic neuron progenitor cells with the markerpolynucleotide probes or antibodies of the present invention; selectingthe dopaminergic neuron progenitor cells; and culturing the selectedcells.

According to the present invention, dopaminergic neurons, which have alow risk of tumorigenesis and are suited to transplant therapy, can alsobe obtained by culturing the dopaminergic neuron progenitor cellsselected by using the marker polynucleotide probes or antibodies of thepresent invention; and selecting and/or screening by using thedopaminergic neuron markers. The dopaminergic neuron markers caninclude, for example, DAT (Development 131(5): 1145-55 (2004)). Forexample, the dopaminergic neurons can be selected and/or produced bycontacting cell samples containing the dopaminergic neuron progenitorcells with the marker polynucleotide probes or antibodies of the presentinvention; selecting the dopaminergic neuron progenitor cells; andcontacting an antibody against DAT with the further cultureddopaminergic neuron progenitor cells to select cells expressing DAT.

The present invention provides methods for selecting and/or producingdopaminergic neuron proliferative progenitor cells capable ofproliferating when cultivated, where the methods comprise the steps ofculturing or not culturing the dopaminergic neuron progenitor cellsselected by using the antibodies of the present invention; and removingthe postmitotic dopaminergic neuron precursor cells. The presentinvention also provides methods for selecting and/or producing thedopaminergic neuron proliferative progenitor cells capable ofproliferating when cultivated, where the methods comprise the steps ofcontacting cell samples containing the dopaminergic neuron progenitorcells with the antibodies of the present invention; selecting thedopaminergic neuron progenitor cells; culturing or not culturing theselected cells; and then removing the postmitotic dopaminergic neuronprecursor cells.

In the step of removing the postmitotic dopaminergic neuron precursorcells, dopaminergic neuron proliferative progenitor cells capable ofproliferating when cultivated can also be obtained by selection and/orremoval using markers for postmitotic dopaminergic neuron precursorcells. Markers for postmitotic dopaminergic neuron precursor cells caninclude, for example, 65B13, Nurrl, and TH (WO 2004/038018; Kawasaki etal., Neuron 28:31-40 (2000); Wallen et al., Exp. Cell Res., 253:737-46(1999)). Dopaminergic neuron proliferative progenitor cells can beselected and/or produced by, for example, contacting the antibodies ofthe present invention with cell samples containing dopaminergic neuronprogenitor cells, selecting the dopaminergic neuron progenitor cells,and contacting an antibody against the 65B13 polypeptide with thedopaminergic neuron progenitor cells further cultured as necessary toselect cells which do not express the 65B13 polypeptide.

In addition, Lrp4-expressing dopaminergic neuron progenitor cells canalso be selected and/or screened using promoters for Lrp4 (see, forexample, Unexamined Published Japanese Patent Application No.2002-51775). For example, a vector harboring a construct that comprisesa gene encoding a detection marker, such as GFP, linked to a promoterregion obtained from analyzing the Lrp4 expression regulatory regions tobe described later, can be transfected into cells. In addition, a geneencoding a marker can also be knocked in at the Lrp4 gene locus. Ineither case, specific cells can be selected by detecting the expressionof a marker gene specific to dopaminergic neuron progenitor cells.

The cell samples used herein preferably comprise cells of the ventralmidbrain region or culture cells containing in vitro differentiateddopaminergic neuron progenitor cells. In vitro differentiation ofdopaminergic neurons can be carried out by known methods using astarting material like cells such as known ES cells, bone marrowinterstitial cells, immortalized neuron-derived cell lines (PublishedJapanese Translation of International Publication No. Hei 8-509215;Published Japanese Translation of International Publication No. Hei11-506930; Published Japanese Translation of International PublicationNo. 2002-522070), or primordial neuron cells (Published JapaneseTranslation of International Publication No. Hei 11-509729). Normally,dopaminergic neurons can be differentiated by co-culturing a tissueobtained from a dopaminergic neuron region of the brain, with asustentacular cell layer derived from neural tissues. Moreover, methodsare also known for deriving dopaminergic neurons from neural tissuesthat do not normally produce dopamine, such as the striatum and cortex(Published Japanese Translation of International Publication No. Hei10-509319). In addition, culturing under hypoxic conditions has beenreported to produce cells containing a greater number of dopaminergicneurons (Published Japanese Translation of International Publication No.2002-530068). ES cells (CCE) can also be induced to differentiate todopaminergic neurons using the 5-stage method (Lee et al. (2000) Nat.Biotech. 18: 675-679, mouse dopaminergic neuron differentiation kit (R&DSystems)). Cell samples used in the selection of dopaminergic neuronprogenitor cells of the present invention may be cell populationsisolated or cultured by any method, including the above-describedmethods.

In addition, the supports used in immobilizing the antibodies of thepresent invention are preferably safe for cells. Examples of suchsupports include synthetic or naturally-occurring organic polymercompounds, inorganic materials such as glass beads, silica gel, alumina,and activated charcoal, and those with surfaces coated with apolysaccharide or synthetic polymer. There are no particular limitationson the form of the support, examples of which include films, fibers,granules, hollow fibers, non-woven fabrics, porous supports, orhoneycombed supports. The contact surface area of the supports can becontrolled by changing their thickness, surface area, width, length,shape, and size in various ways.

<Kits for Treating Neurodegenerative Diseases Comprising DopaminergicNeuron Progenitor Cells, and Methods for Treating NeurodegenerativeDiseases Using Dopaminergic Neuron Progenitor Cells>

The cells acquired using polynucleotide probes and expression of Lrp4mRNA as an index are dopaminergic neuron proliferative progenitor cells.Cells acquired using the antibodies and expression of Lrp4 polypeptidesas an index are dopaminergic neuron progenitor cells. Thus, by usingeither the mRNAs or the polypeptides as indexes, dopaminergic neuronlineage cell populations can be obtained. The progenitor cells obtainedby the methods of the present invention are more preferable fortransplant therapy for diseases related to postural reflex, movement,and reward-associated behaviors, particularly neurodegenerative diseasessuch as Parkinson's disease, schizophrenia, and drug habits (Hynes etal., Cell 80: 95-101 (1995)) in terms of safety, survival rate, andnetwork forming capacity compared to conventional unpurified cellpopulations or dopaminergic neurons into which exogenous genes areintroduced. The cells acquired using Lrp4 expression as an index can beused for transplantation directly or after in vitro proliferation (FIG.13). Such cells are expected to exert therapeutic effects because theyare likely to differentiate and mature in an optimal place in the brain.Therefore, the present invention also provides kits (hereinafter,sometimes referred to as “kits of the present invention”) for treatingneurodegenerative diseases, where the kits comprises dopaminergic neuronproliferative progenitor cells, postmitoticc dopaminergic neuronprecursor cells, and/or dopaminergic neurons selected and/or producedusing the marker polynucleotide probes or antibodies of the presentinvention, as well as methods for treating neurodegenerative diseaseswhich comprise the step of transplanting the cells into the brains ofpatients. Furthermore, the present invention also provides uses ofdopaminergic neuron proliferative progenitor cells, postmitoticdopaminergic neuron precursor cells, and/or dopaminergic neuronsselected and/or produced using the marker polynucleotide probes orantibodies of the present invention for producing the kits for treatingneurodegenerative disease. In the present invention, the term“neurodegenerative diseases” preferably includes Parkinson's disease.The kits of the present invention may comprise pharmaceuticallyacceptable carriers in addition to the cells. The term “carriers”include, for example, salines, phosphate buffers, media, sera, bodyfluids, carboxymethyl cellulose solutions, scaffolding solids forsupporting cells (e.g., cytodex3 (Amersham Bioscience, 17-0485-01)),extracellular matrix components (e.g., collagen, fibronectin,vitronectin, laminin, heparan sulfate, proteoglycan, glucosaminoglycan,chondroitin sulfate, hyaluronate, elastin, or combination of two or morethereof), or gel-like supports. A pH adjuster, buffer, stabilizer,preservative, and the like can be added to the kits of the presentinvention. The kits of the present invention may be for single ormultiple inoculation. It is also possible to appropriately select dosagedepending on the body weight and age of the humans or animals to beinoculated, the administration methods, and so on.

Since the dopaminergic neuron progenitor cells selected using Lrp4expression as an index are likely to further proliferate in vivo, theyare expected to exert therapeutic effects for an extended period.Furthermore, the dopaminergic neuron progenitor cells selected using theLrp4 expression as an index can be differentiated in vitro to anappropriate stage by selecting conditions such as the medium, and arepreferred as the materials for various neural transplant therapies. Asdescribed above, for example, the dopaminergic neuron progenitor cellsselected using Lrp4 expression as an index can be subjected to furtherselection using markers (e.g., 65B13, Nurrl, TH, etc.) for postmitoticdopaminergic neuron precursor cells, to obtain even safer cells for usein transplantation. The present invention relates to methods forculturing the above-described progenitor cells in vitro fordifferentiation and proliferation. The dopaminergic neuron progenitorcells for culturing can be postmitotic precursor cells.

For example, 1×10² to 1×10⁸ dopaminergic neuron progenitor cellsobtained using the methods of the present invention can be transplanted,preferably 1×10³ to 1×10⁶ cells, and more preferably 5×10⁴ to 6×10⁴cells. The primary method is stereotaxic surgery, in which a cellsuspension is transplanted into the brain. In addition, cells may alsobe transplanted by microsurgery. For methods of transplanting neurontissues see Backlund et al. (Backlund et al. (1985) J. Neurosurg. 62:169-73), Lindvall et al. (Lindvall et al. (1987) Ann. Neurol. 22:457-68), or Madrazo et al. (Madrazo et al. (1987) New Engl. J. Med. 316:831-4).

Moreover, the cells of the present invention can also be used to isolategenes specific to dopaminergic neuron proliferative progenitor cells,and genes specific to each stage of maturation from proliferativeprogenitor cells into dopaminergic neurons. They can also be used tosearch for therapeutic targets for Parkinson's disease, to elucidate thematuration process of dopaminergic neurons, and in screenings usingmaturation as an indicator.

<Comparison of Gene Expression Levels>

Dopaminergic neuron progenitor cells, obtained using the polynucleotideprobes and antibodies of the present invention, can be used as materialsto isolate genes specifically expressed in these cells. They can also beused to investigate and isolate genes specifically expressed in cellsthat have been differentiated, induced, or proliferated from thedopaminergic neuron progenitor cells of the present invention. Inaddition, they can also be used to investigate the genes required for invivo differentiation of dopaminergic neurons, by investigating genesthat have different expression levels in cells that have differentiated,induced, or proliferated and the original progenitor cells. Since suchgenes are potential candidates for treating diseases caused by defectsin dopaminergic neurons, determining and isolating them is extremelyuseful.

Comparison of gene expression levels in the dopaminergic neuronprogenitor cells of the present invention with those in cells that havebeen differentiated, induced, or proliferated therefrom, or other cells;or comparison of gene expression levels of the differentiated, induced,or proliferated cells with those of other cells, can be done usingcommonly used methods, such as cell in situ hybridization, Northern blothybridization, RNA dot blot hybridization, reverse transcription PCR,RNase protection assay, DNA microarray hybridization, serial analysis ofgene expression (SAGE) (Velculescu et al. (1995) Science 270: 484-487),subtractive hybridization, and representation difference analysis (RDA)(Lisitsyn (1995) Trends Genet. 11: 303-307).

For cellular in situ hybridization, places where RNA processing,transport, and localization into the cytoplasm occur in individual cellscan be investigated, by hybridizing total RNA or poly A⁺ RNA preparedfrom cells with a labeling probe specific to a given RNA sequence. Inaddition, RNA size can be determined from size fractioning using gelelectrophoresis. Moreover, RNA transcription products can be visualizedin situ by using quantitative fluorescent in situ hybridization (FISH)and a digital imaging microscope (Femino et al. (1998) Science 280:585-90), which are applicable to the present invention.

When using reverse transcription PCR for gene expression analysis, theexpression of specific genes can be roughly quantified. Various isoformsof a single RNA transcription product can also be detected and analyzedusing the present methods. For reverse transcription PCR, when thereaction is carried out using exon-specific primers, and amplificationproducts other than the predicted product are detected, mRNA isoformsproduced by alternative splicing can be identified by analyzing theseproducts. For more details see, for example, the method described inPykett et al. (1994) Hum. Mol. Genet. 3: 559-64. When a quick and roughanalysis of expression pattern is required, the methods which use PCR ofthe present invention are particularly preferred, in terms of their highspeed, high sensitivity, and simplicity.

The efficiency of gene expression screening can be improved by using DNAchips. Herein, a DNA chip refers to a miniature array in whicholigonucleotides, DNA clones, or such are immobilized at a high densityon a support surface, such as glass. For example, in order to carry outmultiple expression screening, cDNA clones for each gene of interest, oroligonucleotides specific to each gene, are immobilized on a chip toproduce a microarray. Next, RNAs are prepared from the dopaminergicneuron progenitor cells of the present invention, or cellsdifferentiated, induced, or proliferated therefrom, and treated withreverse transcriptase to yield cDNAs. Next, the resulting cDNA samplesare labeled with fluorescent tags or other tags, and then hybridized tothe microarray. As a result, genes that are actively expressed in thecells have a higher percentage of total labeled cDNA, while genes thatare not significantly expressed have a lower percentage. Namely, thefluorescent signal intensity, which represents hybridization between alabeled cDNA and a cDNA clone or an oligonucleotide on the chip,reflects the expression level of each sequence in the labeled cDNA, andthereby enables quantification of gene expression.

In addition, multiple genes in the dopaminergic neuron progenitor cellsof the present invention, or cells differentiated, induced, orproliferated therefrom, can be simultaneously analyzed by mRNAdifferential display, which involves reverse transcription PCR usingdegenerate PCR primers. First, a modified oligo dT primer is prepared,in which one or two nucleotides at the 3′ terminus in the poly A tail ofa given mRNA have been altered. Then, a reverse transcription reactionis carried out using the total RNAs isolated from the dopaminergicneuron progenitor cells of the present invention, cells differentiatedor proliferated therefrom, or control cells to be used for expressioncomparison (Liang et al. (1993) Nucleic Acids Res. 21: 3269-3275). Ifthe altered nucleotide is a “G”, then mRNAs with a “C” immediatelybefore the poly A tail can be selectively amplified. If the alterednucleotides are “CA”, then mRNAs with “TG” immediately before the poly Atail can be selectively amplified. Next, an arbitrary nucleotidesequence of about 10 nucleotides in length is prepared for use as asecond primer, and a PCR amplification reaction is carried out using themodified oligo dT primer and this second primer. The amplificationproduct is subjected to size fractionation by electrophoresis using along polyacrylamide gel. By using such methods, cDNAs derived from themRNAs specifically expressed in either of the cells of the presentinvention or the control cells can be detected as bands only present inthe samples that have been electrophoresed. These methods can also beused to analyze expression of unidentified genes.

SAGE analysis does not require a special device for detection, and isone of the preferred analytical methods for simultaneously detecting theexpression of a large number of transcription products. First, poly A⁺RNA is extracted from the dopaminergic neuron progenitor cells of thepresent invention, or cells differentiated, induced, or proliferatedtherefrom, using standard methods. Next, the RNAs are converted intocDNAs using a biotinylated oligo (dT) primer, and are then treated witha four-base recognizing restriction enzyme (Anchoring Enzyme: AE). Here,the AE-treated fragments contain a biotin group at their 3′ terminus.Next, the AE-treated fragments are incubated with streptoavidin forbinding. The bound cDNA is divided into two fractions, and each fractionis then linked to a different double-stranded oligonucleotide adapter(linker) A or B. These linkers are composed of: (1) a protruding singlestrand portion having a sequence complementary to the sequence of theprotruding portion formed by the action of the anchoring enzyme, (2) a5′ nucleotide recognizing sequence of the IIS-type restriction enzyme(cleaves at a predetermined location no more than 20 bp away from therecognition site) serving as a tagging enzyme (TE), and (3) anadditional sequence of sufficient length for constructing a PCR-specificprimer. Herein, the linker-linked cDNA is cleaved using the taggingenzyme, and only the linker-linked cDNA sequence portion remains, whichis present in the form of a short-strand sequence tag. Next, pools ofshort-strand sequence tags from the two different types of linkers arelinked to each other, followed by PCR amplification using primersspecific to linkers A and B. As a result, the amplification product isobtained as a mixture comprising myriad sequences of two adjacentsequence tags (ditags) bound to linkers A and B. The amplificationproduct is treated with the anchoring enzyme, and the free ditagportions are linked into strands in a standard linkage reaction. Theamplification product is then cloned. The clone's determined nucleotidesequence can be used to obtain a read-out of consecutive ditags ofconstant length. The presence of mRNA corresponding to each tag can thenbe identified once from the determination of the clone's nucleotidesequence and information on the sequence tags thus obtained.

Subtraction hybridization is frequently used to clone genes withdifferent expression levels in various tissues or cells, and can also beused to clone genes specifically expressed in the dopaminergic neuronprogenitor cells of the present invention, or cells differentiated,induced, or proliferated therefrom. First, from the dopaminergic neuronprogenitor cells of the present invention, a DNA sample of a cell to betested is prepared (hereinafter referred to as “test DNA”). Next, a DNAof a cell to be compared is prepared (hereinafter referred to as “driverDNA”). The test and driver DNAs can also be used interchangeably. In anycase, genes present in the test DNA but absent from the driver DNA aredetected. Next, the prepared test DNA is mixed with a large excess ofdriver DNA, denatured to form single-stranded DNA, then annealed. Byregulating the annealing conditions, specific sequences absent from thedriver DNA can be isolated as double-stranded DNAs comprising only thetest DNA sequence. For further detail on this method see, Swaroop et al.(1991) Nucleic Acids Res. 19: 1954 and Yasunaga et al. (1999) NatureGenet. 21: 363-9.

The RDA method is a method that uses PCR to selectively amplify asequence of a test DNA that is absent in a driver DNA, and can be usedin the present invention similarly to other previously describedmethods. For more details on the procedure see Lisitsyn (1995) TrendsGenet. 11: 303-7 and Schutte et al. (1995) Proc. Natl. Acad. Sci. USA92: 5950-4.

Genes specific to dopaminergic neuron progenitor cells, or cellsdifferentiated, induced, or proliferated therefrom, are detected andisolated as described, and can be inserted into vectors or such, forsequence determination and expression analysis using the various knownmethods described above.

<Screening Using Dopaminergic Neuron Progenitor Cell Maturation as anIndex>

The present invention provides screening methods that comprise the stepof contacting test substances with the dopaminergic neuron progenitorcells of the present invention, and the step of detecting thedifferentiation or proliferation of the progenitor cells that resultsfrom that contact. Since compounds obtained by this screening methoddemonstrate a regulatory function in the differentiation, proliferation,and such, of dopaminergic neurons, they are considered useful aspotential therapeutic candidates for diseases caused by defects indopaminergic neurons. The dopaminergic neuron progenitor cells of thepresent invention include cells selected by using the polynucleotideprobes or antibodies of the present invention, and cells obtained by theproliferation and/or differentiation induction of these cells.

Here, the “test substance” may be any type of compound, examples ofwhich include the expression products of gene libraries, synthetic lowmolecular weight compound libraries, synthetic peptide libraries,antibodies, substances released by bacteria, cell (microbial, plant, oranimal) extracts, cell (microbial, plant, or animal) culturesupernatants, purified or partially purified polypeptides, marineorganisms, plant or animal extracts, soil, random phage peptide displaylibraries, and such.

Cell differentiation and proliferation can be detected by comparisonwith cell status in the absence of the test substance. Celldifferentiation and proliferation can be detected by morphologicalobservation under a microscope or by detection or quantification ofsubstances produced in cells, such as dopamine.

<Analysis of Lrp4 Expression Regulatory Region>

Using a sequence of the Lrp4 gene, an expression regulatory region ofLrp4 can be cloned from genomic DNA by known methods. For example, amethod for establishing the transcriptional start site, such as the S1mapping method, is known and can be used (Cell Engineering, Supplement8, New Cell Engineering Experiment Protocol, Cancer Research Division,The Institute of Medical Science, The University of Tokyo ed., ShujunshaPublishing (1993) pp. 362-374). In general, the expression regulatoryregion of a gene can be cloned by screening genomic DNA libraries, usingprobe DNAs comprising a 15-100 bp segment, and preferably a 30-50 bpsegment, of the gene's 5′ terminus (in the present invention, all or aportion of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2). A cloneobtained in this manner contains a 5′ non-coding region of 10 kbp ormore, and is shortened or fragmented by exonuclease treatment, or such.Finally, the shortened sequence portion, comprising a potentialexpression regulatory region, is evaluated for the strength, regulationand such of its expression using a reporter gene, thereby making itpossible to determine the minimum unit required to maintain the activityof the Lrp4 expression regulatory region.

Gene expression regulatory regions can be predicted using a program suchas Neural Network (http://www.fiuitfly.org./seg_tools/promoter.html;Reese et al., Biocomputing: Proceedings of the 1996 Pacific Symposium,Hunter and Klein ed., World Scientific Publishing Co., Singapore,(1996)). Moreover, a program for predicting the minimum unit requiredfor the activity of an expression regulatory region is also known andcan be used(http://biosci.cbs.umn.edu./software/proscan/promoterscan.htm;Prestridge (1995) J. Mol. Biol. 249: 923-932).

The expression regulatory region of the Lrp4 gene isolated in thismanner can be used to produce proteins of interest specifically indopaminergic neuron proliferative progenitor cells in vivo.

<Ligand for Lrp4>

The Lrp4 polypeptides have a transmembrane domain, and thus in natureare thought to exist embedded within the cell membrane. Due to itsexpression in dopaminergic neuron progenitor cells, Lrp4 is believed tobe involved in the regulation of dopaminergic neuron progenitor cellproliferation and in neuron differentiation and maturation. Thus,potential ligands that may demonstrate an agonistic or antagonisticfunction towards Lrp4 may be used to regulate the differentiation ofdopaminergic neurons in vivo, ex vivo, and in vitro. In identifyingligands for Lrp4 polypeptides, an Lrp4 polypeptide and a candidatecompound are first contacted and tested for the presence of binding. Inthis case, the Lrp4 polypeptide can be used when immobilized on asupport, or embedded in the cell membrane. There are no particularlimitations on the candidate compounds, examples of which includeexpression products of gene libraries, natural substances derived frommarine organisms, extracts of various types of cells, known compoundsand peptides, natural substances derived from plants, body tissueextracts, microbial culture supernatants and peptide groups randomlyproduced by the phage display method (J. Mol. Biol. 222: 301-10 (1991)).In addition, the candidate compound may be labeled for detection ofbinding.

<Inhibition of Lrp4 Expression>

Since the present invention clearly demonstrates that Lrp4 mRNA istransiently expressed in dopaminergic neuron proliferative progenitorcells, Lrp4 may be involved in the control of progenitor cellproliferation as well as neuron differentiation and maturation. Thus,substances that inhibit Lrp4 gene expression may be used to control thedifferentiation of dopaminergic neurons in vivo, ex vivo, and in vitro.Examples of substances capable of inhibiting gene expression includeantisense nucleic acids, ribozymes, and double-stranded RNAs (smallinterfering RNA; siRNA). Thus, the present invention provides suchantisense nucleic acids, ribozymes, and double-stranded RNAs.

Examples of antisense mechanisms that suppress target gene expressioninclude: (1) inhibition of transcription initiation via triplexformation, (2) transcription suppression through hybrid formation atsites of local open-loop structures formed by RNA polymerase, (3)transcription inhibition through hybrid formation with RNA duringsynthesis, (4) suppression of splicing through hybrid formation atintron-exon junctions, (5) suppression of splicing through hybridformation at sites of spliceosome formation, (6) suppression of mRNAmigration to the cytoplasm through hybrid formation with mRNA, (7)suppression of splicing through hybrid formation at a capping site orpoly A addition site, (8) suppression of translation initiation throughhybrid formation at binding sites of translation initiation factors, (9)translation suppression through hybrid formation at ribosome bindingsites, (10) suppression of peptide chain elongation through hybridformation at mRNA coding regions or polysome binding sites, and (11)suppression of gene expression through hybrid formation at sites ofnucleic acid/protein interaction (Hirashima and Inoue, “New BiochemistryExperiment Course 2, Nucleic Acids IV, Gene Replication and Expression”,Japanese Biochemical Society edit., Tokyo Kagaku Dozin Publishing, pp.319-347 (1993)).

An Lrp4 antisense nucleic acid of the present invention may be a nucleicacid that inhibits gene expression by any of the mechanisms described in(1) to (11) above. Namely, it may contain an antisense sequence to notonly a sequence of a coding region, but also a sequence of a non-codingregion of a target gene whose expression is to be inhibited. A DNA thatencodes an antisense nucleic acid can be used by linking to a suitableregulatory sequence that allows its expression. The antisense nucleicacid does not need to be completely complementary to the coding regionor non-coding region of a target gene, as long as it can effectivelyinhibit the expression of this gene. Such antisense nucleic acids have achain length of at least 15 bp or more, preferably 100 bp or more, andmore preferably 500 bp or more, and are normally within 3000 bp,preferably within 2000 bp, and more preferably within 1000 bp. It ispreferable that such antisense nucleic acids share an identity of 90% ormore, and more preferably 95% or more, with the complementary chain of atarget gene transcription product. These antisense nucleic acids can beprepared according to phosphorothionate methods (Stein (1988) NucleicAcids Res. 16: 3209-21) or the like, using Lrp4 polynucleotides.

“Ribozyme” is a generic term referring to catalysts with an RNAcomponent, and ribozymes are broadly classified into large ribozymes andsmall ribozymes. Large ribozymes cleave the phosphate-ester bonds of anucleic acid, and after reaction, they leave 5′-phosphoric acid and3′-hydroxyl group at the reaction sites. Large ribozymes are furtherclassified into (1) group I intron RNAs, which carry outguanosine-initiated trans-esterification reactions at 5′-splice sites,(2) group II intron RNAs, which perform two-step self-splicing reactionsvia a lariat structure, and (3) RNA components of ribonuclease P, whichcleave precursor tRNAs at their 5′ side via hydrolysis reactions. Incontrast, small ribozymes are comparatively small structural units(about 40 bp) that cleave RNAs, forming 5′-hydroxyl groups and 2′-3′cyclic phosphoric acids. Small ribozymes include, for example,hammerhead-type ribozymes (Koizumi et al. (1988) FEBS Lett. 228: 225)and hairpin-type ribozymes (Buzayan (1986) Nature 323: 349; Kikuchi andSasaki (1992) Nucleic Acids Res. 19: 6751; Kikuchi (1992) Chemistry andBiology 30: 112). Since ribozymes are easily altered and synthesized,various methods for their modification are known. For example,hammerhead-type ribozymes that recognize and cleave nucleotide sequenceUC, UU, or UA within a target RNA can be created, by designing thesubstrate binding portion of a ribozyme to be complementary to an RNAsequence near the target site (Koizumi et al. (1988) FEBS Lett. 228:225; M. Koizumi and E. Ohtsuka (1990) Protein, Nucleic Acid and Enzyme35: 2191; Koizumi et al. (1989) Nucleic Acids Res. 17: 7059).Hairpin-type ribozymes can also be designed and produced using knownmethods (Kikuchi and Sasaki (1992) Nucleic Acids Res. 19: 6751; Kikuchi(1992) Chemistry and Biology 30: 112).

Antisense nucleic acids and ribozymes of the present invention can alsobe used in viral vectors derived from retroviruses, adenoviruses,adeno-associated viruses, and such, or non-viral vectors that useliposomes, or naked DNAs, to control gene expression in cells using exvivo or in vivo gene therapy.

RNA interference is a phenomenon in which the introduction of anartificial double-stranded RNA into cells causes RNAs having the samenucleotide sequence to be degraded (Fire et al. (1998) Nature 391:806-11). There are no particular limitations on the siRNAs of thepresent invention, provided they inhibit transcription of Lrp4 mRNA.Normally, an siRNA is a combination of a sense and antisense chain tothe sequence of a target mRNA, and has a nucleotide length of at least10 to the same number of nucleotides as the target mRNA. These siRNAspreferably have a nucleotide length of 15 to 75, preferably 18 to 50,and more preferably 20 to 25 nucleotides.

In order to suppress Lrp4 expression, siRNAs can be introduced intocells using known methods. For example, a DNA is designed to encode, ina single strand, two RNA chains that compose an siRNA, and this is thenincorporated into an expression vector, cells are transformed with theexpression vector, and the siRNA can be expressed in the cells in theform of a double-stranded RNA with a hairpin structure. Plasmidexpression vectors that continuously produce siRNA by transfection havealso been designed (for example, RNAi-Ready pSIREN Vector, andRNAi-Ready pSIREN-RetroQ Vector (BD Biosciences Clontech)).

The nucleotide sequence of an siRNA can be designed using a computerprogram such as that disclosed at the Ambion website(http://www.ambion.coni/techlib/misc/siRNA_finder.html). Kits forscreening for functional siRNAs are also commercially available and canbe used (for example, BD Knockout RNAi System (BD Biosciences Clontech).

EXAMPLES

The present invention will be specifically described using Examples, butit is not be construed as being limited thereto.

Example 1 Isolation and Sequence Analysis of a Gene Specific toDopaminergic Neuron Progenitor Cells

To isolate genes specific to dopaminergic neuron progenitor cells, themidbrain ventral region of E12.5 mice was additionally cut into tworegions in the dorsoventral direction, and genes specifically expressedin the most ventral region containing dopaminergic neurons wereidentified by the subtraction (N-RDA) method. One of the isolated cDNAfragments was a fragment encoding Lrp4/Corin. Lrp4 encodes type IItransmembrane proteins (FIG. 1).

(1) N-RDA Method (1)-1. Adapter Preparation

The following oligonucleotides were annealed to each other, and preparedat 100 μM.

(ad2: ad2S + ad2A, ad3: ad3S + ad3A, ad4: ad4S + ad4A, ad5: ad5S + ad5A,ad13: ad13S + ad13A) ad2S: cagctccacaacctacatcattccgt (SEQ ID NO: 5)ad2A: acggaatgatgt (SEQ ID NO: 6) ad3S: gtccatcttctctctgagactctggt (SEQID NO: 7) ad3A: accagagtctca (SEQ ID NO: 8) ad4S:ctgatgggtgtcttctgtgagtgtgt (SEQ ID NO: 9) ad4A: acacactcacag (SEQ ID NO:10) ad5S: ccagcatcgagaatcagtgtgacagt (SEQ ID NO: 11) ad5A: actgtcacactg(SEQ ID NO: 12) ad13S: gtcgatgaacttcgactgtcgatcgt (SEQ ID NO: 13) ad13A:acgatcgacagt (SEQ ID NO: 14)(1)-2. cDNA Synthesis

Ventral midbrain regions were cut out of E12.5 mouse embryos (JapanSLC), and divided into two sections in the dorsoventral direction. TotalRNA was prepared using the RNeasy Mini Kit (Qiagen), and double-strandedcDNA was synthesized using a cDNA Synthesis Kit (Takara). Afterdigestion with restriction enzyme RsaI, ad2 was added. The cDNA wasamplified by a 5-minute incubation at 72° C., 15 PCR cycles of 30seconds at 94° C., 30 seconds at 65° C., and 2 minutes at 72° C., and afinal 2-minute incubation at 72° C. using ad2S as the primer. In allcases, N-RDA PCR was carried out using a reaction solution containingthe following components.

10×ExTaq 5 μl

2.5 mM dNTP 4 μl

ExTaq 0.25 μl

100 μM primer 0.5 μl

cDNA 2 μl

Distilled water 38.25 μl

(1)-3. Driver Production

The ad2S-amplified cDNA was further amplified by incubating at 94° C.for 2 minutes, and then performing five PCR cycles of 30 seconds at 94°C., 30 seconds at 65° C., and 2 minutes at 72° C., and a final 2-minuteincubation at 72° C. The cDNA was purified using the Qiaquick PCRPurification Kit (Qiagen), and digested with RsaI. 3 μg was used foreach round of subtraction.

(1)-4. Tester Production

The ad2S amplified cDNA was further amplified by incubating at 94° C.for 2 minutes, and then performing five PCR cycles of 30 seconds at 94°C., 30 seconds at 65° C., and 2 minutes at 72° C., and a final 2-minuteincubation at 72° C. The cDNA was purified using the Qiaquick PCRPurification Kit (Qiagen), and digested with RsaI. ad3 was added to 60ng of the RsaI-digested cDNA.

(1)-5. First Round of Subtraction

The tester and the driver produced in Sections 1-3 and 1-4 above weremixed, subjected to ethanol precipitation, and then dissolved in 1 μl of1×PCR buffer. After a 5-minute incubation at 98° C., 1 μl of 1×PCRbuffer+1M NaCl was added. After another 5 minutes of incubation at 98°C., the tester and the driver were hybridized at 68° C. for 16 hours.

With ad3S as the primer, the hybridized cDNA was amplified by incubatingat 72° C. for 5 minutes, and performing ten cycles of 30 seconds at 94°C., 30 seconds at 65° C., and 2 minutes at 72° C. Next, the amplifiedcDNA was digested with the Mung Bean Nuclease (Takara) and purifiedusing a Qiaquick PCR Purification Kit. Then, it was amplified byincubating at 94° C. for 2 minutes, and performing 13 PCR cycles of 30seconds at 94° C., 30 seconds at 65° C., and 2 minutes at 72° C., and afinal 2-minute incubation at 72° C.

(1)-6. Normalization

1 μL of 2×PCR buffer was added to 8 ng of the cDNA amplified in thefirst round of subtraction. After incubating at 98° C. for 5 minutes, 2μl of 1×PCR buffer+1 M NaCl was added. After another 5 minutes ofincubation at 98° C., the cDNA was hybridized at 68° C. for 16 hours.

The hybridized cDNA was digested with RsaI, and purified using theQiaquick PCR Purification Kit. Then, it was amplified with ad3S as theprimer by incubating at 94° C. for 2 minutes, and performing 11 PCRcycles of 30 seconds at 94° C., 30 seconds at 65° C., and 2 minutes at72° C., and a final 2-minute incubation at 72° C. The PCR product wasthen digested with RsaI, followed by the addition of ad4.

(1)-7. Second Round of Subtraction

20 ng of the cDNA to which ad4 was added in Section 1-6 above was usedas the tester and mixed with the driver of 1-3 above, and the samesubtraction procedure used in Section 1-5 above was performed. Finally,ad5 was added to the cDNA following RsaI digestion.

(1)-8. Third Round of Subtraction

2 ng of the cDNA to which ad5 was added in section 1-7 above was used asthe tester and mixed with the driver of 1-3 above, and the samesubtraction procedure used in section 1-5 above was carried out.Finally, ad13 was added to the RsaI-digested cDNA.

(1)-9. Fourth Round of Subtraction

2 ng of the cDNA to which ad13 was added in section 1-8 above was usedas the tester and mixed with the driver of 1-3 above, and the samesubtraction procedure used in Section 1-5 above was carried out. Theamplified cDNA was cloned into pCRII vector (Invitrogen), and itsnucleotide sequence was analyzed using the ABI3100 sequence analyzer.

Example 2 Expression Analysis of the Lrp4 Gene

Next, an expression analysis of the Lrp4 gene by in situ hybridizationwas carried out according to the following protocol.

First, E12.5 mouse embryos were embedded in O.C.T., and fresh frozensections of 16 μm thickness were prepared. After drying on a slideglass, the sections were fixed in 4% PFA at room temperature for 30minutes. After washing with PBS, hybridization was carried out at 65° C.for 40 hours (1 μg/ml DIG-labeled RNA probe, 50% formamide, 5×SSC, 1%SDS, 50 μg/ml yeast RNA, 50 μg/ml Heparin). Subsequently, the sectionswere washed at 65° C. (50% formamide, 5×SSC, 1% SDS) and then treatedwith RNase (5 μg/ml RNase) at room temperature for 5 minutes. Afterwashing with 0.2×SSC at 65° C. and washing with 1×TBST at roomtemperature, blocking was carried out (Blocking reagent: Roche). Thesections were then reacted with alkaline phosphatase-labeled anti-DIGantibody (DAKO), washed (1×TBST, 2 mM Levamisole), and color developedusing NBT/BCIP (DAKO) as the substrate.

The expression analysis by in situ hybridization showed that Lrp4 mRNAis specifically expressed in the ventral midline region from themidbrain to the hindbrain and the spinal cord at the stage E12.5, whichcorresponds to the time of dopaminergic neuron development. Lrp4demonstrates a similar expression pattern to Shh mRNA from the hindbrainto the spinal cord, and was clearly determined to be specific to thefloor plate, which is the organizer region (FIGS. 2 and 5). In themidbrain, Lrp4 expression was observed more centrally than the Shh mRNAexpression zone (FIGS. 3 and 5).

The results of comparison with the neuron maturation marker NCAM mRNAshow that Lrp4 mRNA-expressing cells were proliferative progenitor cellsin the NCAM mRNA-negative ventricular zone (VZ). Moreover, when comparedwith the expression of the dopaminergic neuron marker, TH mRNA, theirexpression regions completely overlapped along the dorsal-ventral axis(FIGS. 3 and 5), although expression of both TH mRNA and Lrp4 mRNA inthe same cells was not observed since TH mRNA is only expressed in themantle layer (ML). In general, neurons present in neural tubes are knownto first proliferate in the VZ, exit cell cycle with the commencement ofdifferentiation, and then mature after migrating to the outer ML. Thus,dopaminergic neuron proliferative progenitor cells are believed toproliferate in the VZ which lines the TH expression zone, and express THmRNA after having migrated to the outside following cell cycle exit.Namely, Lrp4 mRNA is believed to be specifically expressed in themidbrain in dopaminergic neuron proliferative progenitor cells (FIGS. 4and 6).

Example 3 Expression of Lrp4 in Dopaminergic Neurons Induced toDifferentiate from ES Cells

Next, whether Lrp4 is expressed in ES cells that have been induced todifferentiate into dopaminergic neurons in vitro, was examined.

First, dopaminergic neurons were induced to differentiate from ES cellsusing the SDIA method (Kawasaki et al. (2000) Neuron 28(1): 3140) (seethe upper part of FIG. 7). Cells were recovered 4, 6, 8, 10, and 12 daysafter induction, and total RNA was recovered using the RNeasy Mini Kit(Qiagen) followed by RT-PCR. In RT-PCR, cDNA was initially synthesizedfor 1 μg of total RNA using the RNA PCR Kit (TaKaRa). PCR was thencarried out in the following reaction system, using as a template cDNAequivalent to 10 ng, 1 ng, and 0.1 ng.

10x ExTaq 2 μl 2.5 mM dNTP 1.6 μl ExTaq 0.1 μl 100 μM primer 0.2 μl eachcDNA 1 μl Distilled water 14.9 μl

After incubating for 2 minutes at 94° C., 35 PCR cycles of 30 seconds at94° C., 30 seconds at 65° C., and 2 minutes at 72° C. were carried outfollowed by incubating for 2 minutes at 72° C.

The sequences of the primers used are shown below.

Lrp4: TAGTCTACCACTGCTCGACTGTAACG/ (SEQ ID NO: 15)CAGAGTGAACCCAGTGGACATATCTG (SEQ ID NO: 16) TH:GTTCCCAAGGAAAGTGTCAGAGTTGG/ (SEQ ID NO: 17) GAAGCTGGAAAGCCTCCAGGTGTTCC(SEQ ID NO: 18) DAT: CTCCGAGCAGACACCATGACCTTAGC/ (SEQ ID NO: 19)AGGAGTAGGGCTTGTCTCCCAACCTG (SEQ ID NO: 20)

According to the results of expression analysis by RT-PCR, although Lrp4is not expressed in ES cells (CCE) or stroma cells (PA6), expression wasclearly induced starting on day 4 in the same manner as TH as a resultof inducing differentiation (FIG. 8). Thus, the marker polynucleotideprobes of the present invention are useful as markers not only whenisolating dopaminergic neuron proliferative progenitor cells from thefetal midbrain, but also when isolating dopaminergic neuronproliferative progenitor cells that have been induced to differentiatefrom ES cells in vitro.

Example 4 Analysis of Lrp4 Protein Expression

The anti-Lrp4 antibody was produced by the protocol described belowusing the extracellular region encoding sequence in the Lrp4 gene, andexpression analyses were performed using immunohistological staining.

First, the extracellular region (161 to 502 amino acids) encodingsequence in the Lrp4 gene was introduced into 293E cells, and theextracellular region of the Lrp4 protein was expressed and collected.Hamsters were immunized with the collected protein, and lymphocytes wereremoved therefrom and fused with myeloma cells. The fused cells werecultured to obtain a culture supernatant. On day 12.5 mouse embryos werethen fixed with 4% PFA/PBS(−) at 4° C. for two hours, then the solutionwas replaced with 20% sucrose/PBS(−) at 4° C. overnight, and the embryoswere embedded with OTC. 12-μm-thick sections were made, attached on aslide glass, dried at room temperature for 30 minutes, and wetted withPBS(−) again. The sections were blocked (10% normal donkey serum and 10%normal goat serum/Block Ace) at room temperature for 20 minutes, andthen reacted with the anti-Lrp4 monoclonal antibody produced asdescribed above (mixture of FERM BP-10315 and FERM BP-10316 [each¼-diluted culture supernatants, 10% normal donkey serum, 10% normal goatserum, and 2.5% Block Ace/PBS]) and the anti-TH antibody (Chemicon, 0.7μg/mL, 10% normal donkey serum, 10% normal goat serum, and 2.5% BlockAce/PBS) at room temperature for one hour, and further reacted at 4° C.overnight. The sections were washed four times with 0.1% TritonX-100/PBS(−) at room temperature for 10 minutes. Cy3-labeledanti-hamster IgG antibody or the FITC-labeled anti-mouse IgG antibody(Jackson, 10 μg/mL, 10% normal donkey serum, 10% normal goat serum, and2.5% Block Ace/PBS) was reacted with the sections at room temperaturefor one hour. Then, the sections were washed as described above, thenwashed with PBS(−) at room temperature for 10 minutes, and sealed.

As a result of expression analysis by immunohistological staining usingthe produced anti-Lrp4 monoclonal antibody, expression of the Lrp4protein was observed in the ventral midbrain at E12.5, the stage whenthe dopaminergic neuron developed (FIG. 8) as shown by expressionanalysis using in situ hybridization. Compared with expression of the THprotein, which was the dopaminergic neuron marker, the Lrp4 protein wasexpressed in the most ventral midbrain (VZ side) where the TH proteinwas expressed. Accordingly, it appeared that the Lrp4 protein isexpressed in the dopaminergic neuron progenitor cells.

Next, using the anti-Lrp4 monoclonal antibody, Lrp4-expressing cellswere detected by flow cytometry.

First, ES cells were induced to differentiate to dopaminergic neuronprogenitor cells in vitro using the SDIA method. A cell populationcomprising the cells was dispersed using a cell dissociation buffer(Invitrogen), and stained with the anti-Lrp4 monoclonal antibody(mixture of FERM BP-10315 and FERM BP-10316 [each ¼-diluted culturesupernatant, 1% fetal calf serum, and 1 mM EDTA/SDIA differentiationmedium]) at 4° C. for 20 minutes without fixing and permeabilization.Subsequently, the cells were washed three times with 1% fetal calf serumand 1 mM EDTA/SDIA differentiation medium at 4° C. for 3 minutes. Thecells were stained with biotin-labeled anti-hamster IgG antibody(Jackson, 10 μg/mL, 1% fetal calf serum and 1 mM EDTA/SDIAdifferentiation medium) at 4° C. for 20 minutes, and washed as describedabove. Then, the cells were stained with PE-labeled streptavidin(Pharmingen, 20 μg/mL, 1% fetal calf serum and 1 mM EDTA/SDIAdifferentiation medium) at 4° C. for 20 minutes, and washed as describedabove. After staining, the Lrp4-expressing cells were detected by a flowcytometer.

The results of detecting the Lrp4-expressing cells by flow cytometryusing the produced anti-Lrp4 monoclonal antibody detected an Lrp4protein-expressing population (FIG. 9). Since the Lrp4protein-expressing cells could be detected without fixing andpermeabilization, it appeared that living Lrp4 protein-expressing cellscan be separated by using a flow cytometer equipped with a cell sorter.The Lrp4 protein is thought to be expressed in the dopaminergic neuronprogenitor cells, and thus the anti-Lrp4 antibody appeared to be usefulfor the separation of the dopaminergic neuron progenitor cells.

Example 5 Separation of Lrp4-Expressing Cells by Antibodies

Lrp4 protein-positive cells separated using the anti-Lrp4 antibody werecharacterized.

First, a cell population comprising E12.5 mouse fetus ventral midbrainand dopaminergic neuron progenitor cells, induced in vitro todifferentiate from ES cells by the SDIA method, was stained with theanti-Lrp4 antibody by the method described in Example 4. Lrp4-positiveand -negative cells were separated using a cell sorter. Total RNA wascollected from the cells immediately after separation using an RNeasymini kit (Qiagen). Then, cDNA was synthesized, and amplified by the samemethod as described in Example 1 to use as a template in RT-PCR. The PCRwas performed using cDNAs corresponding to the amplified cDNA equivalentto 4 ng, 0.4 ng, and 0.04 ng by the following reaction system.

10 x ExTaq 1 μL 2.5 mM dNTP 0.8 μL ExTaq 0.05 μL 100 μM primers 0.1 μLeach cDNA 1 μL Distilled water 6.95 μL

PCR was carried out under conditions of 94° C. for 2 minutes, 26 cyclesof 94° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 2minutes, and finally 72° C. for 2 minutes.

The sequences of the primers used are shown below.

Lrp4: TAGTCTACCACTGCTCGACTGTAACG/ (SEQ ID NO: 15)CAGAGTGAACCCAGTGGACATATCTG (SEQ ID NO: 16) TH:GTTCCCAAGGAAAGTGTCAGAGTTGG/ (SEQ ID NO: 17) GAAGCTGGAAAGCCTCCAGGTGTTCC(SEQ ID NO: 18) Nurr1: CACTCCTGTGTCTAGCTGCCAGATGC/ (SEQ ID NO: 21)AGTGCGAACACCGTAGTGCTGACAGG (SEQ ID NO: 22) Nestin:GATGAAGAAGAAGGAGCAGAGTCAGG/ (SEQ ID NO: 23) ATTCACTTGCTCTGACTCCAGGTTGG(SEQ ID NO: 24) MAP2: CCATGATCTTTCCCCTCTGGCTTCTG/ (SEQ ID NO: 25)TTTGGCTGGAAAGGGTGACTCTGAGG (SEQ ID NO: 26)

As expected, the results of expression analysis by the RT-PCR indicatedexpression of the proliferative progenitor cell marker Nestin. It wasalso revealed that cells expressing MAP2, which was the postmitoticneuron marker, were included in the Lrp4 protein-positive cellpopulation (FIG. 10). Therefore, it was found that Lrp4 proteinexpression is maintained after stopping mRNA expression, and that Lrp4protein is useful as a marker for separating not only dopaminergicneuron proliferative progenitor cells but also postmitotic dopaminergicneuron precursor cells (FIG. 11). Furthermore, since Nurrl and TH, whichare markers for postmitotic dopaminergic neuron precursor cells, wereexpressed at higher levels compared to the Lrp4-negative cellpopulation, Lrp4-positive cells were confirmed to be dopaminergic neuronlineage progenitor cells (FIG. 10).

Next, the present inventors analyzed the ratio of proliferativeprogenitor cells to postmitotic precursor cells in the Lrp4protein-positive cell population separated by the anti-Lrp4 antibody.

The separated cells were seeded on a glass slide coated withpoly-L-ornithine (Sigma, 0.002% in PBS), laminin (Invitrogen, 5 μg/mL inPBS), and fibronectin (Sigma, 5 μg/mL in PBS), and incubated in N2(Invitrogen, 1×), B27 (Invitrogen 1×), ascorbic acid (Sigma, 200 μM),and BDNF (Invitrogen, 20 ng/mL)/SDIA differentiation medium at 37° C.for 40 minutes to adhere thereon. The adherent cells were fixed in 4%PFA/PBS at 4° C. for 20 minutes, and washed twice with PBS at 4° C. for10 minutes. Permeabilization with 0.3% Triton X-100/PBS was performed atroom temperature for 15 minutes, and blocking with 10% normal donkeyserum/Block Ace was performed at room temperature for 20 minutes. Then,the cells were reacted with anti-Nestin antibody (Chemicon, 2 μg/mL, 10%normal donkey serum, 2.5% Block Ace, 0.1% Triton X-100/PBS) oranti-βIII-tubulin antibody (BABCO, 1/2000, 0.5 μg/mL, 10% normal donkeyserum, 2.5% Block Ace, and 0.1% Triton X-100/PBS) at room temperaturefor one hour, and subsequently at 4° C. overnight. On the next day, thecells were washed three times with 0.1% Triton X-100/PBS at roomtemperature for 5 minutes, and reacted with the FITC-labeled anti-mouseIgG antibody or the Cy5-labeled anti-rabbit IgG antibody (both fromJackson, 10 μg/mL, 10% normal donkey serum, 2.5% Block Ace, and 0.1%Triton X-100/PBS) at room temperature for 30 minutes. Subsequently, thecells were washed as described above, then washed with PBS at roomtemperature for 5 minutes, and sealed for observation.

Also, the separated cells were similarly seeded on a glass slide, andcultured in the above-described medium supplemented with BrdU (Roche,5-Bromo-2′-deoxy-uridine Labeling and Detection kit II, 1×) at 37° C.for 18 hours. Then, the above-described procedures were similarlycarried out until the blocking step. The cells were reacted in 2 N HClat 37° C. for 20 minutes, washed three times with PBS, and then reactedwith anti-BrdU antibody and DNase (Roche, 5-Bromo-2′-deoxy-uridineLabeling and Detection kit II, 1×conc. in incubation buffer) at 37° C.for 30 minutes. Furthermore, the cells were reacted with anti-BrdUantibody (Sigma, 44 μg/mL, 10% normal donkey serum, 2.5% Block Ace, and0.1% Triton X-100/PBS) at room temperature for one hour, andsubsequently at 4° C. overnight. The next day, the cells were washedthree times with 0.1% Triton X-100/PBS at room temperature for 5minutes, and then reacted with the FITC-labeled anti-mouse IgG antibody(Jackson, 10 μg/mL, 10% normal donkey serum, 2.5% Block Ace, and 0.1%Triton X-100/PBS) at room temperature for 30 minutes. Subsequently, thecells were washed, and sealed for observation as described above.

As a result of the marker staining, it was revealed that a majority ofthe Lrp4-positive cells are Nestin-positive proliferative progenitorcells and that a part thereof is positive for the postmitotic markerβIII-tubulin (FIG. 12). In addition, the separated cells were confirmedto frequently incorporate BrdU, and to actually proliferate in vitro(FIG. 13).

Next, the present inventors confirmed that the separated Lrp4-positivecells differentiate into the dopaminergic neurons.

The separated cells were seeded on a glass slide coated withpoly-L-ornithine (Sigma, 0.002% in PBS), laminin (Invitrogen, 5 μg/mL inPBS), and fibronectin (Sigma, 5 μg/mL in PBS), and incubated in N2(Invitrogen, 1×), B27 (Invitrogen 1×), ascorbic acid (Sigma, 200 μM),BDNF (Invitrogen, 20 ng/mL), and bFGF (R&D, 10 ng/ml)/SDIAdifferentiation medium at 37° C. for 24 hours. The cells were thenfurther cultured in the above medium without bFGF for another six days.The cultured cells were fixed in 4% PFA/PBS at 4° C. for 20 minutes, andwashed twice with PBS at 4° C. for 10 minutes. Permeabilization with0.3% Triton X-100/PBS was performed at room temperature for 15 minutes,and blocking with 10% normal donkey serum/Block Ace was performed atroom temperature for 20 minutes. Then, the cells were reacted withanti-TH antibody (Chemicon, 0.3 μg/mL, 10% normal donkey serum, 2.5%Block Ace, 0.1% Triton X-100/PBS) or anti-βIII-tubulin antibody (BABCO,1/2000, 0.5 μg/mL, 10% normal donkey serum, 2.5% Block Ace, and 0.1%Triton X-100/PBS) at room temperature for one hour, and subsequently at4° C. overnight. On the next day, the cells were washed three times with0.1% Triton X-100/PBS at room temperature for 5 minutes, and reactedwith the FITC-labeled anti-mouse IgG antibody or the Cy5-labeledanti-rabbit IgG antibody (both from Jackson, 10 μg/mL, 10% normal donkeyserum, 2.5% Block Ace, and 0.1% Triton X-100/PBS) at room temperaturefor 30 minutes. Subsequently, the cells were washed as described above,then washed with PBS at room temperature for 5 minutes, and sealed forobservation.

As a result of culturing the separated cells in vitro, it was obviousthat more TH protein-positive dopaminergic neurons were induced thanwith the unseparated control cells. Therefore, it was revealed that theLrp4-positive cells are certainly dopaminergic neuron lineage progenitorcells and can mature in vitro (FIG. 14).

Example 6 Transplantation of Separated Lrp4-Positive Cells into Striatumin Parkinson's Disease Model Mice

Lrp4-expressing cells were separated by flow cytometry using anti-Lrp4monoclonal antibody, and the cells were transplanted into the striatumof Parkinson's disease model mice.

First, ES cells were induced to differentiate to dopaminergic neuronprogenitor cells in vitro using the SDIA method. A cell populationcomprising the cells was dispersed using a cell dissociation buffer(Invitrogen), and stained with the anti-Lrp4 monoclonal antibodyprepared in Example 4 (mixture of FERM BP-10315 and FERM BP-10316 [each¼-diluted culture supernatant, 1% fetal calf serum, and 1 mM EDTA/SDIAdifferentiation medium]) at 4° C. for 20 minutes without fixing andpermeabilization. Subsequently, the cells were washed three times with1% fetal calf serum and 1 mM EDTA/SDIA differentiation medium at 4° C.for 3 minutes. The cells were stained with biotin-labeled anti-hamsterIgG antibody (Jackson, 10 μg/mL, 1% fetal calf serum and 1 mM EDTA/SDIAdifferentiation medium) at 4° C. for 20 minutes, and washed as describedabove. Then, the cells were stained with PE-labeled streptavidin(Pharmingen, 20 μg/mL, 1% fetal calf serum and 1 mM EDTA/SDIAdifferentiation medium) at 4° C. for 20 minutes, and washed as describedabove. After staining, the Lrp4-expressing cells were separated by aflow cytometer.

Next, the present inventors transplanted the separated Lrp4protein-positive cells into the striatum of Parkinson's disease modelmice, and characterized the Lrp4 protein-positive cells in the brain.

First, the Parkinson's disease model mice were prepared by injecting1.25 μL of 6-OHDA (Sigma, 2 μg/μL) into one side of the medial forebrainbundle of 12-week-old mice (slc) to deaden the dopaminergic neuronsprojecting from the midbrain to the striatum. Two weeks after preparingthe model mice, 3×10⁴ Lrp4-positive cells per mouse were transplantedinto the striatum to which 6-OHDA had been injected. The Lrp4protein-positive cells to be transplanted were obtained by: transfectingES cells to express the EGFP gene under the control of CAG promoter(Niwa et al., Gene 108:193-200 (1991)); inducing the ES cells todifferentiate into the dopaminergic neuron progenitor cells in vitro bythe SDIA method; and staining the cell population comprising thedopaminergic neuron progenitor cells with the anti-Lrp4 antibody asdescribed in Example 4 and separating by the cell sorter.

Three weeks after the transplantation, 500 μL of 10% urethan in salinewas intraperitoneally injected to anesthetize the mice. Underanesthesia, thoracotomy was performed, 30 mL of saline (Otsuka) wasinjected from left ventricle for perfusion, and then perfusion fixationwas carried out with 30 mL of 4% PFA/PBS(−). After fixation, the brainswere removed, and further immersion-fixed in 4% PFA/PBS(−) for anothereight hours. The brains were then sliced at a thickness of 2 mm, thesolution was replaced with 20 to 40% sucrose/PBS(−) overnight, and thebrains were embedded in OCT. 10 to 12 μm-thick sections were made,attached on glass slides, dried at room temperature for 30 minutes, andwetted again with PBS(−). Blocking (10% normal donkey serum/Block Ace)was then performed at room temperature for 20 minutes. The sections werethen reacted with anti-GFP antibody (Molecular probes, 20 μg/mL, 10%normal donkey serum, and 10% Block Ace/PBS), anti-MAP2 antibody (Sigma,mouse ascites, diluted 100 times, 10% normal donkey serum, and 10% BlockAce/PBS), or anti-TH antibody (Chemicon, 1 μg-mL, 10% normal donkeyserum and 10% Block Ace/PBS) at room temperature for one hour, and againat 4° C. overnight. Sections were washed four times with 0.1% TritonX-100/PBS(−) at room temperature for 10 minutes. The sections were thenreacted with Alexa Flour 488-labeled anti-rabbit IgG antibody (MolecularProbes, 4 μg/mL, 10% normal donkey serum, and 10% Block Ace/PBS),Cy3-labeled anti-mouse IgG antibody (Jackson, 10 μg/mL, 10% normaldonkey serum, and 10% Block Ace/PBS), or Cy5-labeled anti-sheep IgGantibody (Jackson, 10 μg/mL, 10% normal donkey serum, and 10% BlockAce/PBS) at room temperature for one hour. Then, the sections werewashed as described above, washed with PBS(−) at room temperature for 10minutes, and sealed.

The expression of various markers was analyzed by immunohistologicalstaining.

As a result, EGFP-positive cells were observed in the striatum of thetransplanted mice (Table 1). This suggests that the transplanted Lrp4protein-positive cells are successfully engrafted in the striatum of theParkinson's disease model mice.

Most of the successfully engrafted cells were also observed to bepositive for the mature neuron marker MAP2, and EGFP-positive axons wereobserved to extend far into the striatum (Table 1 and FIG. 16).

TABLE 1 EGFP+ TH+ CELLS CELLS TH+ #6 SEQ ID NO: 20 95 19 20% #7 SEQ IDNO: 12 131 21 16%(Table 1 shows the in vivo differentiation of the transplantedLrp4-positive cells into TH-positive cells. Two weeks after deadeningthe dopaminergic neurons with 6-OHDA, the Lrp4 protein-positive cellswere transplanted into mice #6 and #7. Perfusion was performed threeweeks after transplant.

The results indicated that the transplanted Lrp4 protein-positive cellsare nerve progenitor cells, whereas most successfully engrafted cellsdifferentiate and mature into mature nerve cells. About 20% of thesuccessfully engrafted cells were TH-positive, strongly suggesting thatat least a part of the transplanted Lrp4 protein-positive cellsdifferentiate into dopaminergic neurons.

Therefore, the dopaminergic neuron progenitor cells separated accordingto the present invention are able to differentiate into the dopaminergicneurons by being transplanted into the brain, and thus the dopaminergicneuron progenitor cells separated according to the present invention arebelieved useful for therapies.

Example 7 Lrp4 Expression in Dopaminergic Neurons Induced toDifferentiate from ES Cells Using the 5-Stage Method

The present inventors examined whether or not Lrp4 is expressed when EScells are induced to differentiate into the dopaminergic neurons invitro using the 5-stage method.

First, ES cells (CCE) were induced to differentiate into dopaminergicneurons using the 5-stage method (Lee et al., Nat. Biotech., 18:675-679(2000), mouse dopaminergic neuron differentiation kit [R&D Systems]).Cells were collected on day 2 at Stage 1, day 4 at Stage 2, day 6 atStage 3, days 4 and 6 at Stage 4, and days 4 and 7 at Stage 5. TotalRNAs were prepared using RNeasy mini kit (Qiagen) to perform RT-PCR. Inthe RT-PCR, cDNA was first synthesized from 1 μg of the total RNA usingRNA PCR kit (TaKaRa). The PCR was performed by the following reactionsystem, using cDNAs corresponding to 10 ng, 1 ng, and 0.1 ng astemplates.

10 x ExTaq 2 μL 2.5 mM dNTP 1.6 μL ExTaq 0.1 μL 100 μM primers 0.2 μLeach cDNA 1 μL Distilled water 14.9 μL

PCR was carried out under conditions of 94° C. for 2 minutes, 32 cyclesof 94° C. for 30 seconds, 65° C. for 30 seconds, and 72° C. for 2minutes, and finally 72° C. for 2 minutes.

The sequences of the primers used are shown below.

Lrp4: TAGTCTACCACTGCTCGACTGTAACG/ (SEQ ID NO: 15)CAGAGTGAACCCAGTGGACATATCTG (SEQ ID NO: 16) TH:GTTCCCAAGGAAAGTGTCAGAGTTGG/ (SEQ ID NO: 17) GAAGCTGGAAAGCCTCCAGGTGTTCC(SEQ ID NO: 18)

The results of expression analysis by RT-PCR revealed that Lrp4 is notexpressed in Stage 1, at which the cells are undifferentiated ES cells,but is induced to be expressed in Stage 4, at which the dopaminergicneuron progenitor cells are developed (FIG. 17A).

Next, the present inventors detected the Lrp4-expressing cells by flowcytometry using the anti-Lrp4 monoclonal antibody when ES cells wereinduced to differentiate into the dopaminergic neurons in vitro usingthe 5-stage method.

First, the cell population (day 7 at Stage 4) comprising thedopaminergic neuron progenitor cells induced to differentiate from EScells in vitro using the 5-stage method was dispersed using the celldispersing solution Accumax (Innovative Cell Technologies, Inc.).Without the steps of fixing and permeabilization, the cells were stainedwith anti-Lrp4 monoclonal antibody (mixture of FERM BP-10315 and FERMBP-10316 [each ½-diluted culture supernatant]) at 4° C. for 20 minutes.The cells were then washed three times with 1% fetal calf serum, 1 mMEGTA, 4.5 mg/mL glucose, and 40 ng/mL DNase I/Ca- and Mg-free Hanks'balanced salt solution (HBSS-) at 4° C. for 3 minutes. The cells werestained with the PE-labeled anti-hamster IgG antibody (8 μg/mL [BDBiosciences], 1% fetal calf serum, 1 mM EGTA, 4.5 mg/mL glucose, and 40ng/mL DNase I/Ca- and Mg-free Hanks' balanced salt solution (HBSS-)) at4° C. for 30 minutes, and washed as described above. After staining,Lrp4-expressing cells were detected using a flow cytometer.

The results of detecting Lrp4-expressing cells by flow cytometry usinganti-Lrp4 monoclonal antibody detected an Lrp4 protein-expressingpopulation in the cell group comprising the dopaminergic neuronprogenitor cells induced to differentiate from the ES cells in vitrousing the 5-stage method (FIG. 17B). This suggested that Lrp4 isexpressed in the dopaminergic neuron progenitor cells induced todifferentiate from the ES cells by not only the SDIA method but also bythe 5-stage method, and is useful as a marker for cell separation.

Example 8 Differentiation and Maturation in Vitro of Lrp4-ExpressingCells Separated by Antibodies

The present inventors examined whether or not Lrp4 protein-positivecells separated using anti-Lrp4 antibody differentiate into dopaminergicneurons in vitro.

The cell population comprising the dopaminergic neuron progenitor cellsinduced to differentiate from ES cells in vitro using the 5-stage methodwas stained using anti-Lrp4 antibody by the method described in Example4. The Lrp4-positive cells were then separated using a cell sorter. Theseparated cells were seeded on a glass slide coated withpoly-L-ornithine (Sigma, 0.002% in PBS) and fibronectin (Sigma, 5 μg/mLin PBS), and incubated in N2 (Invitrogen, 1×), ascorbic acid (Sigma, 200μM), and BDNF (R&D Systems, 20 ng/mL)/DMEM/F12 at 37° C. for 7 days. Thecultured cells were fixed in 2% PFA and 0.15% picric acid/PBS at 4° C.for 20 minutes, and washed twice with PBS at 4° C. for 10 minutes.Permeabilization with 0.3% Triton X-100/PBS was performed at roomtemperature for 30 minutes, and blocking with 10% normal donkey serumand 10% normal goat serum/Block Ace was performed at room temperaturefor 20 minutes. Then, the cells were reacted with anti-TH antibody(Chemicon, 0.4 μg/mL, 10% normal donkey serum, 10% normal goat serum,2.5% Block Ace, 0.1% Triton X-100/PBS) or anti-MAP2 antibody (Sigma,1/200 ascites, 0.5 μg/mL, 10% normal donkey serum, 10% normal goatserum, 2.5% Block Ace, and 0.1% Triton X-100/PBS) at room temperaturefor one hour, and subsequently at 4° C. overnight. On the next day, thecells were washed four times with 0.1% Triton X-100/PBS at roomtemperature for 10 minutes, and reacted with the FITC-labeled anti-mouseIgG antibody or the Cy3-labeled anti-rabbit IgG antibody (both fromJackson, 10 μg/mL, 10% normal donkey serum, 10% normal goat serum, 2.5%BlockAce, and 0.1% Triton X-100/PBS) at room temperature for 30 minutes.Subsequently, the cells were washed as described above, then washed withPBS at room temperature for 5 minutes, and sealed for observation.

Culturing the separated Lrp4-positive cells in vitro induced many THprotein-positive dopaminergic neurons (FIG. 17C). Therefore, it wasrevealed that Lrp4-positive cells induced by the 5-stage method aredopaminergic neuron progenitor cells, and can mature in vitro. The Lrp4was expressed in the dopaminergic neuron progenitor cells induced by twodifferent differentiation methods (SDIA method and 5-stage method), anddopaminergic neuron progenitor cells induced by either method could beseparated using the anti-Lrp4 antibody. Accordingly, it appeared thatLrp4 is useful as a marker for dopaminergic neuron progenitor cellsderived from any cell source. The 5-stage method is capable of inducingdifferentiation of dopaminergic neuron progenitor cells without contactwith animal-derived cells and components, and expected to be appliedclinically. Lrp4 is useful as a cell separation marker in the methods,and thus appeared to be most likely applied to transplant therapy forneurodegenerative diseases including Parkinson's disease.

Example 9 Elimination of Undifferentiated ES Cells by SeparatingDopaminergic Neuron Progenitor Cells Using Anti-Lrp4 Antibody

When ES cell-derived transplant cells are prepared, it is most importantin terms of safety to eliminate the undifferentiated ES cells thatresult in teratoma Since Lrp4 is not expressed in undifferentiated EScells (Example 3), separation using Lrp4 as a marker is expected toeliminate undifferentiated ES cells. To confirm this hypothesis, thepresent inventors used RT-PCR to examine the expression of ERas (Nature,423(6939):541-5 (2003)) and Nanog (Cell, 113(5):631-42 (2003)), whichare ES cell-specific genes, to investigate whether undifferentiated EScells were contained in cells separated using anti-Lrp4 antibody fromcells induced to differentiate from ES cells in vitro using the SDIAmethod.

First, using the method described in Example 5, Lrp4-positive and-negative cells were separated by a cell sorter from a cell populationcomprising dopaminergic neuron progenitor cells induced to differentiatefrom ES cells in vitro using the SDIA method. Total RNA was collectedimmediately after separation, and the cDNA to be amplified was prepared.PCR was performed using cDNAs corresponding to 4 ng, 0.4 ng, and 0.04 ngas templates for the following reaction system:

10x ExTaq 1 μL 2.5 mM dNTP 0.8 μL ExTaq 0.05 μL 100 μM primers 0.1 μLeach cDNA 1 μL Distilled water 6.95 μL

PCR was carried out under conditions of 94° C. for 2 minutes, 26 cycles(for Lrp4 and Nestin) or 30 cycles (for ERas and Nanog) of 94° C. for 30seconds, 65° C. for 30 seconds, and 72° C. for 2 minutes, and finally72° C. for 2 minutes.

The sequences of the primers used are shown below (the sequences of Lrp4and Nestin were described in Example 5).

ERas: TGCTCTCACCATCCAGATGACTCACC/ (SEQ ID NO: 27)TGGACCATATCTGCTGCAACTGGTCC (SEQ ID NO: 28) Nanog:TCCAGCAGATGCAAGAACTCTCCTCC/ (SEQ ID NO: 29) TTATGGAGCGGAGCAGCATTCCAAGG(SEQ ID NO: 30)

As a result, ERas and Nanog, which are expressed specifically in EScells, were not expressed in the Lrp4-positive cell population; on theother hand, ERas and Nanog expressions were detected in theLrp4-negative cell population (FIG. 18). Accordingly, it was revealedthat undifferentiated ES cells contained in the cells induced todifferentiate by the SDIA method could be eliminated by cell separationusing Lrp4.

INDUSTRIAL APPLICABILITY

The present invention identified Lrp4 as a gene expressed specificallyand transiently in dopaminergic neuron proliferative progenitor cells.As a result of examining Lrp4 expression in more detail, it wasconfirmed that Lrp4 mRNA and the Lrp4 protein were expressedspecifically in dopaminergic neuron proliferative progenitor cells andin dopaminergic neuron progenitor cells, including cells prior to andafter cell cycle exit, respectively. Thus, by using the expression ofLrp4 mRNA or an Lrp4 polypeptide in the cells as an index, it becamepossible in terms of safety, survival rate, and network formation toselect dopaminergic neuron lineage cells suitable for transplant therapyfor neurodegenerative diseases including Parkinson's disease. When thecells are obtained using Lrp4 as a marker, as in the present invention,the cells can be easily differentiated into a suitable state in vitroeven when the therapy requires mature cells. Moreover, dopaminergicneuron progenitor cells obtained by the methods of the present inventioncan also be used to isolate genes specifically expressed in these cells.The cells are also thought to be useful in developing pharmaceuticalsfor neurodegenerative diseases such as Parkinson's disease. Sincedopaminergic neuron proliferative progenitor cells obtained using Lrp4mRNA as a marker are involved in early neuron formation, they are usefulin elucidating the neuron maturation process, namely, for identifyingvarious factors involved in the maturation process. Elucidation of thesefactors is expected to contribute greatly to the treatment ofneurodegenerative diseases. Moreover, maturation of these cells can beused as an index for screening substances that may regulate (inhibit orpromote) the maturation process.

1. A dopaminergic neuron proliferative progenitor cell markerpolynucleotide probe comprising a sequence selected from the followingnucleotide sequences (1) to (5): (1) a nucleotide sequence complementaryto a nucleotide sequence of SEQ ID NO: 1 or 2; (2) a nucleotide sequencecomplementary to a nucleotide sequence encoding an amino acid sequenceof SEQ ID NO: 3 or 4; (3) a nucleotide sequence complementary to anucleotide sequence encoding a sequence lacking a transmembrane domainin an amino acid sequence of SEQ ID NO: 3 or 4; (4) a nucleotidesequence that hybridizes under stringent conditions with apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or 2;and, (5) a nucleotide sequence comprising at least 15 contiguousnucleotides selected from sequences of (1) to (4).
 2. A method forselecting a dopaminergic neuron proliferative progenitor cell, whereinthe method comprises the step of contacting the polynucleotide probe ofclaim 1 with a cell sample thought to comprise a dopaminergic neuronproliferative progenitor cell.
 3. A method for selecting a dopaminergicneuron lineage cell, wherein the method comprises the steps of: (1)selecting a dopaminergic neuron proliferative progenitor cell using themethod of claim 2 for selecting the dopaminergic neuron proliferativeprogenitor cell; (2) culturing the proliferative progenitor cellselected in step (1); and (3) screening the cells cultured in step (2)by using a marker for a postmitotic dopaminergic neuron.
 4. Adopaminergic neuron proliferative progenitor cell, which is selected bythe method of claim
 2. 5. A method for isolating a dopaminergic neuronproliferative progenitor cell-specific gene and a gene specific for eachmaturation stage from the proliferative progenitor cell to adopaminergic neuron, wherein the method comprises the step of detectingand isolating a gene specifically expressed in the proliferativeprogenitor cell of claim 4 or a cell which is differentiated, induced,or proliferated from the proliferative progenitor cell.
 6. A method ofscreening for a compound which regulates proliferation and/ordifferentiation of a dopaminergic neuron lineage cell using maturationas an index, wherein the method comprises the steps of: contacting atest substance with the proliferative progenitor cell of claim 4 or acell which is differentiated, induced, or proliferated from theproliferative progenitor cell; and detecting a change of theproliferative progenitor cell or the progenitor cell caused by thecontact.
 7. An antibody against a polypeptide selected from thefollowing (1) to (6): (1) a polypeptide encoded by a polynucleotideconsisting of a nucleotide sequence of SEQ ID NO: 1 or 2; (2) apolypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4; (3)a polypeptide comprising an amino acid sequence lacking a transmembranedomain in an amino acid sequence of SEQ ID NO: 3 or 4; (4) a polypeptidecomprising an amino acid sequence with a deletion, insertion,substitution, or addition of one or more amino acids in an amino acidsequence of SEQ ID NO: 3 or 4; (5) a polypeptide encoded by apolynucleotide that hybridizes under stringent conditions with apolynucleotide consisting of a sequence complementary to a nucleotidesequence of SEQ ID NO: 1 or 2; and, (6) a polypeptide that is a fragmentof a polypeptide of (1) to (5) comprising at least eight amino acidresidues.
 8. The antibody of claim 7, which is produced by the hybridomaFERM BP-10315 or FERM BP-10316.
 9. A dopaminergic neuron progenitor cellmarker antibody, which comprises the antibody of claim
 7. 10. A methodfor selecting a dopaminergic neuron progenitor cell, wherein the methodcomprises the step of contacting the antibody of claim 7 with a cellsample thought to comprise a dopaminergic neuron progenitor cell.
 11. Amethod for selecting a dopaminergic neuron lineage cell, wherein themethod comprises the steps of: (1) selecting a dopaminergic neuronproliferative progenitor cell using the method of claim 10; (2)culturing the progenitor cell selected in step (1); and (3) screeningthe progenitor cells cultured in step (2) by using a marker for apostmitotic dopaminergic neuron.
 12. A dopaminergic neuron progenitorcell, which is selected by the method of claim
 10. 13. A method forisolating a dopaminergic neuron progenitor cell-specific gene and a genespecific for each maturation stage from the progenitor cell to adopaminergic neuron, wherein the method comprises the step of detectingand isolating a gene specifically expressed in the progenitor cell ofclaim 12 or a cell which is differentiated, induced, or proliferatedfrom the progenitor cell.
 14. A method of screening for a compound whichregulates proliferation and/or differentiation of a dopaminergic neuronlineage cell using maturation as an index, wherein the method comprisesthe steps of: contacting a test substance with the progenitor cell ofclaim 12 or a cell which is differentiated, induced, or proliferatedfrom the progenitor cell; and detecting a differentiated or proliferatedprogenitor cell caused by the contact.
 15. A kit for treatingParkinson's disease, which comprises the dopaminergic neuronproliferative progenitor cell of claim 4 or the dopaminergic neuronprogenitor cell of claim
 12. 16. A method for treating Parkinson'sdisease, wherein the method comprises the step of transplanting thedopaminergic neuron proliferative progenitor cell of claim 4 or thedopaminergic neuron progenitor cell of claim 12 into the brain of apatient.
 17. A use of the dopaminergic neuron proliferative progenitorcell of claim 4 or the dopaminergic neuron progenitor cell of claim 12,for producing a kit for treating Parkinson's disease.
 18. A method fordetecting or selecting a dopaminergic neuron proliferative progenitorcell, which comprises the step of contacting a cell sample comprisingthe dopaminergic neuron proliferative progenitor cell with a secondpolynucleotide which hybridizes under stringent conditions with a firstpolynucleotide consisting of any one of: (1) the nucleotide sequence ofSEQ ID NO: 1 or 2; (2) a nucleotide sequence consisting of apolynucleotide encoding a polypeptide consisting of the amino acidsequence of SEQ ID NO: 3 or 4; (3) a nucleotide sequence consisting of apolynucleotide encoding a polypeptide consisting of an amino acidsequence which lacks a transmembrane region in the amino acid sequenceof SEQ ID NO: 3 or 4; and (4) a nucleotide sequence consisting of apolynucleotide which hybridizes with a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 1 or 2 under stringent conditions. 19.The method of claim 18, wherein the second polynucleotide comprises atleast 15 nucleotides.
 20. A dopaminergic neuron proliferative progenitorcell population, which is selected by the method of claim
 18. 21. Areagent for discriminating a dopaminergic neuron proliferativeprogenitor cell, which comprises a second polynucleotide as an activeingredient which hybridizes under stringent conditions with a firstpolynucleotide consisting of any one of: (1) the nucleotide sequence ofSEQ ID NO: 1 or 2; (2) a nucleotide sequence consisting of apolynucleotide encoding a polypeptide consisting of the amino acidsequence of SEQ ID NO: 3 or 4; (3) a nucleotide sequence consisting of apolynucleotide encoding a polypeptide comprising an amino acid sequencewhich lacks a transmembrane region in the amino acid sequence of SEQ IDNO: 3 or 4; and (4) a nucleotide sequence consisting of a polynucleotidewhich hybridizes with a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 1 or 2 under stringent conditions.
 22. The methodof claim 21, wherein the second polynucleotide comprises at least 15nucleotides.
 23. A method for producing a postmitotic dopaminergicneuron precursor cell, wherein the method comprises the steps of: (1)selecting a dopaminergic neuron proliferative progenitor cell by themethod of claim 18; (2) culturing the cell selected in step (1); and (3)selecting the postmitotic dopaminergic neuron precursor cell from thecells cultured in step (2).
 24. A method for producing a dopaminergicneuron, wherein the method comprises the steps of: (1) selecting adopaminergic neuron proliferative progenitor cell by the method of claim18; and (2) culturing the cell selected in step (1).
 25. The method ofclaim 24, further comprising the step of: (3) selecting a dopaminergicneuron from the cells cultured in step (2).
 26. A method for detectingor selecting a dopaminergic neuron progenitor cell, which comprises thestep of contacting a cell sample comprising the dopaminergic neuronprogenitor cell with an antibody which is bound to a polypeptideconsisting of the amino acid sequence of any one of: (1) the amino acidsequence of SEQ ID NO: 3 or 4; (2) an amino acid sequence which lacks atransmembrane region in the amino acid sequence of SEQ ID NO: 3 or 4;(3) an amino acid sequence mutated by one or more amino acid deletions,substitutions, or additions, or any combination thereof, in the aminoacid sequence of SEQ ID NO:3 or 4; and (4) an amino acid sequenceconsisting of a polypeptide encoded by a polynucleotide which hybridizesunder stringent conditions with a polynucleotide consisting of anucleotide sequence complementary to a nucleotide sequence of SEQ ID NO:1 or 2, or a partial sequence thereof.
 27. The method of claim 26,wherein the polypeptide comprising the partial sequence comprises atleast six consecutive amino acid residues.
 28. A dopaminergic neuronprogenitor cell population, which is selected by the method of claim 26.29. The cell population of claim 28, which comprises 40% or moredopaminergic neuron progenitor cells in the entire cells.
 30. A reagentfor discriminating a dopaminergic neuron progenitor cell, whichcomprises an antibody as an active ingredient which is bound to apolypeptide consisting of the amino acid sequence of any one of: (1) theamino acid sequence of SEQ ID NO: 3 or 4; (2) an amino acid sequencewhich lacks a transmembrane region in the amino acid sequence of SEQ IDNO: 3 or 4; (3) an amino acid sequence mutated by one or more amino aciddeletions, substitutions, or additions, or any combination thereof, inthe amino acid sequence of SEQ ID NO:3 or 4; and (4) an amino acidsequence consisting of a polypeptide encoded by a polynucleotide whichhybridizes under stringent conditions with a polynucleotide consistingof a nucleotide sequence complementary to a nucleotide sequence of SEQID NO: 1 or 2, or a partial sequence thereof.
 31. The reagent of claim30, wherein the polypeptide consisting of the partial sequence comprisesat least six consecutive amino acid residues.
 32. The reagent of claim30, wherein the antibody is produced by the hybridoma FERM BP-10315 orFERM BP-10316.
 33. An antibody produced by the hybridoma FERM BP-10315or FERM BP-10316.
 34. A method for producing a dopaminergic neuronproliferative progenitor cell, wherein the method comprises the stepsof: (1) selecting a dopaminergic neuron progenitor cell by the method ofclaim 26; and (2) removing a postmitotic dopaminergic neuron precursorcell to select the dopaminergic neuron proliferative progenitor cell.35. A method for producing a postmitotic dopaminergic neuron precursorcell, wherein the method comprises the steps of: (1) selecting adopaminergic neuron progenitor cell by the method of claim 26; and (2)culturing the cell selected in step (1).
 36. The method of claim 35,further comprising the step of: (3) selecting a postmitotic dopaminergicneuron precursor cell from the cells cultured in step (2).
 37. A methodfor producing a dopaminergic neuron, wherein the method comprises thesteps of: (1) selecting a dopaminergic neuron progenitor cell by themethod of claim 26; and (2) culturing the cell selected in step (1). 38.The method of claim 37, further comprising the step of: (3) selecting adopaminergic neuron from the cells cultured in step (2).
 39. A kit fortreating a neurodegenerative disease, which comprises at least one cellselected from the group consisting of: (1) the dopaminergic neuronproliferative progenitor cell population of claim 20; (2) a postmitoticdopaminergic neuron precursor cell produced by the method of claim 23;(3) a dopaminergic neuron produced by the method of claim 24; (4) adopaminergic neuron produced by the method of claim 25; (5) thedopaminergic neuron progenitor cell population of claim 28; (6) thedopaminergic neuron progenitor cell population of claim 29; (7) adopaminergic neuron proliferative progenitor cell produced by the methodof claim 34; (8) a postmitotic dopaminergic neuron precursor cellproduced by the method of claim 35; (9) a postmitotic dopaminergicneuron precursor cell produced by the method of claim 36; (10) adopaminergic neuron produced by the method of claim 37; and (11) adopaminergic neuron produced by the method of claim
 38. 40. The kit ofclaim 39, wherein the neurodegenerative disease is Parkinson's disease.41. A method for treating a neurodegenerative disease, which comprisesthe step of transplanting into the brain of a patient at least one cellselected from the group consisting of: (1) the dopaminergic neuronproliferative progenitor cell population of claim 20; (2) a postmitoticdopaminergic neuron precursor cell produced by the method of claim 23;(3) a dopaminergic neuron produced by the method of claim 24; (4) adopaminergic neuron produced by the method of claim 25; (5) thedopaminergic neuron progenitor cell population of claim 28; (6) thedopaminergic neuron progenitor cell population of claim 29; (7) adopaminergic neuron proliferative progenitor cell produced by the methodof claim 34; (8) a postmitotic dopaminergic neuron precursor cellproduced by the method of claim 35; (9) a postmitotic dopaminergicneuron precursor cell produced by the method of claim 36; (10) adopaminergic neuron produced by the method of claim 37; and (11) adopaminergic neuron produced by the method of claim
 38. 42. The methodof claim 41, wherein the neurodegenerative disease is Parkinson'sdisease.
 43. A use of at least one cell for producing a kit for treatinga neurodegenerative disease, wherein the cell is selected from the groupconsisting of: (1) the dopaminergic neuron proliferative progenitor cellpopulation of claim 20; (2) a postmitotic dopaminergic neuron precursorcell produced by the method of claim 23; (3) a dopaminergic neuronproduced by the method of claim 24; (4) a dopaminergic neuron producedby the method of claim 25; (5) the dopaminergic neuron progenitor cellpopulation of claim 28; (6) the dopaminergic neuron progenitor cellpopulation of claim 29; (7) a dopaminergic neuron proliferativeprogenitor cell produced by the method of claim 34; (8) a postmitoticdopaminergic neuron precursor cell produced by the method of claim 35;(9) a postmitotic dopaminergic neuron precursor cell produced by themethod of claim 36; (10) a dopaminergic neuron produced by the method ofclaim 37; and (11) a dopaminergic neuron produced by the method of claim38.
 44. The use of claim 43, wherein the neurodegenerative disease isParkinson's disease.